Picobiology : Life Science at the atomic level

Picobiology: Life Science at the atomic level" was selected in 2007 as one of the 13 programs in the field, "Life Science


1)  Publications




Shinya Yoshikawa, Kazumasa Muramoto, and Kyoko Shinzawa-Itoh

Proton-Pumping Mechanism of Cytochrome c Oxidase

Annual Review of Biophysics, 40, 205-223 (2011)


Cytochrome c oxidase (CcO), as the terminal oxidase of cellular respiration, coupled with a proton-pumping process, reduces molecular oxygen (O2) to water. This intriguing and highly organized chemical process represents one of the most critical aspects of cellular respiration. It employs transition metals (Fe and Cu) at the O2 reduction site and has been considered one of the most challenging research subjects in life science. Extensive X-ray structural and mutational analyses have provided two different proposals with regard to the mechanism of proton pumping. One mechanism is based on bovine CcO and includes an independent pathway for the pumped protons. The second mechanistic proposal includes a common pathway for the pumped and chemical protons and is based upon bacterial CcO. Here, recent progress in experimental evaluations of these proposals is reviewed and strategies for improving our understanding of the mechanism of this physiologically important process are discussed.

Yasufumi Umena Keisuke Kawakami Jian-Ren Shen & Nobuo Kamiya

Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å

Nature, 473, 55-60 (2011)


Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 Å. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 Å. From our electron density map, we located all of the metal atoms of the Mn4CaO5 cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn4CaO5 cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.


Koichi Sakamoto, Masakatsu Kamiy, Mizue Imai, Kyoko Shinzawa-Itoh, Takeshi Uchida, Keiichi Kawano, Shinya Yoshikawa and Koichiro Ishimori

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

Proc Natl Acad Sci U S A. (2011)


The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by 1H-15N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field 1H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by 1H- and 1H, 15N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.


Keisuke Saito, Toyokazu Ishida, Miwa Sugiura, Keisuke Kawakami, Yasufumi Umena, Nobuo Kamiya, Jian-Ren Shen, and Hiroshi Ishikita

Distribution of the Cationic State over the Chlorophyll Pair of the Photosystem II Reaction Center

J. Am. Chem. Soc., 133, 14379–14388 (2011)


The reaction center chlorophylls a (Chla) of photosystem II (PSII) are composed of six Chla molecules including the special pair Chla PD1/PD2 harbored by the D1/D2 heterodimer. They serve as the ultimate electron abstractors for water oxidation in the oxygen-evolving Mn4CaO5 cluster. Using the PSII crystal structure analyzed at 1.9 Å resolution, the redox potentials of PD1/PD2 for one-electron oxidation (Em) were calculated by considering all PSII subunits and the protonation pattern of all titratable residues. The Em(Chla) values were calculated to be 1015–1132 mV for PD1 and 1141–1201 mV for PD2, depending on the protonation state of the Mn4CaO5 cluster. The results showed that Em(PD1) was lower than Em(PD2), favoring localization of the charge of the cationic state more on PD1. The PD1•+/PD2•+ charge ratio determined by the large-scale QM/MM calculations with the explicit PSII protein environment yielded a PD1•+/PD2•+ ratio of 80/20, which was found to be due to the asymmetry in electrostatic characters of several conserved D1/D2 residue pairs that cause the Em(PD1)/Em(PD2) difference, e.g., D1-Asn181/D2-Arg180, D1-Asn298/D2-Arg294, D1-Asp61/D2-His61, D1-Glu189/D2-Phe188, and D1-Asp170/D2-Phe169. The larger PD1•+ population than PD2•+ appears to be an inevitable fate of the intact PSII that possesses water oxidation activity.


Hideto Matsuoka, Jian-Ren Shen, Asako Kawamori, Kei Nishiyama, Yasunori Ohba, and Seigo Yamauchi

Proton-Coupled Electron-Transfer Processes in Photosystem II Probed by Highly Resolved g-Anisotropy of Redox-Active Tyrosine YZ

J. Am. Chem. Soc., 2011, 133 4655–4660 (2011)


The oxidation of a redox-active tyrosine residue YZ in photosystem II (PSII) is coupled with proton transfer to a hydrogen-bonded D1-His190 residue. Because of the apparent proximity of YZ to the water-oxidizing complex and its redox activity, it is believed that YZ plays a significant role in water oxidation in PSII. We investigated the g-anisotropy of the tyrosine radical YZ• to provide insight into the mechanism of YZ• proton-coupled electron transfer in Mn-depleted PSII. The anisotropy was highly resolved by electron paramagnetic resonance spectroscopy at the W-band (94.9 GHz) using PSII single crystals. The gX-component along the phenolic C−O bond of YZ• was calculated by density functional theory (DFT). It was concluded from the highly resolved g-anisotropy that YZ loses a phenol proton to D1-His190 upon tyrosine oxidation, and D1-His190 redonates the same proton back to YZ• upon reduction.


Masato Murakami, Dachao Hong, Tomoyoshi Suenobu, Satoru Yamaguchi, Takashi Ogura and Shunichi Fukuzumi

Catalytic Mechanism of Water Oxidation with Single-Site Ruthenium-Heteropolytungstate Complexes

J. Am. Chem. Soc., 2011, 133, 11605–1161.


Catalytic water oxidation to generate oxygen was achieved using all-inorganic mononuclear ruthenium complexes bearing Keggin-type lacunary heteropolytungstate, [RuIII(H2O)SiW11O39]5– (1) and [RuIII(H2O)GeW11O39]5– (2), as catalysts with (NH4)2[CeIV(NO3)6] (CAN) as a one-electron oxidant in water. The oxygen atoms of evolved oxygen come from water as confirmed by isotope-labeled experiments. Cyclic voltammetric measurements of 1 and 2 at various pH’s indicate that both complexes 1 and 2 exhibit three one-electron redox couples based on ruthenium center. The Pourbaix diagrams (plots of E1/2 vs pH) support that the Ru(III) complexes are oxidized to the Ru(V)–oxo complexes with CAN. The Ru(V)–oxo complex derived from 1 was detected by UV–visible absorption, EPR, and resonance Raman measurements in situ as an active species during the water oxidation reaction. This indicates that the Ru(V)–oxo complex is involved in the rate-determining step of the catalytic cycle of water oxidation. The overall catalytic mechanism of water oxidation was revealed on the basis of the kinetic analysis and detection of the catalytic intermediates. Complex 2 exhibited a higher catalytic reactivity for the water oxidation with CAN than did complex 1.


Takahiko Kojima, Kazuya Nakayama, Kenichiro Ikemura, Takashi Ogura and Shunichi Fukuzumi

Formation of a Ruthenium(IV)-Oxo Complex by Electron-Transfer Oxidation of a Coordinatively Saturated Ruthenium(II) Complex and Detection of Oxygen-Rebound Intermediates in C-H Bond Oxygenation

J. Am. Chem. Soc., 2011, 133,11692–11700.


A coordinatively saturated ruthenium(II) complex having tetradentate tris(2-pyridylmethyl)amine (TPA) and bidentate 2,2′-bipyridine (bpy), [Ru(TPA)(bpy)]2+ (1), was oxidized by a Ce(IV) ion in H2O to afford a Ru(IV)-oxo complex, [Ru(O)(H+TPA)(bpy)]3+ (2). The crystal structure of the Ru(IV)-oxo complex 2 was determined by X-ray crystallography. In 2, the TPA ligand partially dissociates to be in a facial tridentate fashion and the uncoordinated pyridine moiety is protonated. The spin state of 2, which showed paramagnetically shifted NMR signals in the range of 60 to −20 ppm, was determined to be an intermediate spin (S = 1) by the Evans’ method with 1H NMR spectroscopy in acetone-d6. The reaction of 2 with various oraganic substrates in acetonitrile at room temperature afforded oxidized and oxygenated products and a solvent-bound complex, [Ru(H+TPA)(bpy)(CH3CN)], which is intact in the presence of alcohols. The oxygenation reaction of saturated C–H bonds with 2 proceeds by two-step processes: the hydrogen abstraction with 2, followed by the dissociation of the alcohol products from the oxygen-rebound complexes, Ru(III)-alkoxo complexes, which were successfully detected by ESI-MS spectrometry. The kinetic isotope effects in the first step for the reaction of dihydroanthrathene (DHA) and cumene with 2 were determined to be 49 and 12, respectively. The second-order rate constants of C–H oxygenation in the first step exhibited a linear correlation with bond dissociation energies of the C–H bond cleavage.


Nobutaka Fujieda, Takuya Ikeda, Michiaki Murata, Sachiko Yanagisawa, Shigetoshi Aono, Kei Okubo, Satoshi Nagao, Takashi Ogura, Shun Hirota, Shunichi Fukuzumi, Yukihiro Nakamura, Yoji Hata, and Shinobu Itoh,

Post-translational His-Cys Cross Linkage Formation in Tyrosinase Induced by Copper(II)-Peroxo Species, J. Am. Chem. Soc.,133, 1180-1183 (2011).


Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with CuII under aerobic conditions, where a (μ-η2:η2-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.


Ryo Nagao, Akira Moriguchi, Tatsuya Tomo, Ayako Niikura, Saori Nakajima, Takehiro Suzuki, Akinori Okumura, Masako Iwai, Jian-Ren Shen, Masahiko Ikeuchi, and Isao Enami

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom, Chaetoceros gracilis

J. Biol. Chem. 285: 29191-29199 (2010)


Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl and Ca2+ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl and Ca2+ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.



Hashimoto M, Shinohara K, Wang J, Ikeuchi S, Yoshiba S, Meno C, Nonaka S, Takada S, Hatta K, Wynshaw-Boris A, Hamada H.

Planar polarization of node cells determines the rotational axis of the node cilia

Nature Cell Biology 12, 170-176 (2010)


Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo because the cilia are posteriorly tilted. However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a–/– embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.


Muramoto K, Ohta K, Shinzawa-Itoh K, Kanda K, Taniguchi M, Nabekura H, Yamashita E, Tsukihara T, Yoshikawa S.

Bovine Cytochrome c Oxidase Structures Enable O2 Reduction with Minimization of Active Oxygen Species and Provide a Proton Pumping Gate

Proc Natl Acad Sci U S A. 107, 7740-7745 (2010)


The O2 reduction site of cytochrome c oxidase (CcO), comprising iron (Fea3) and copper (CuB) ions, is probed by X-ray structural analyses of CO, NO and CN- derivatives to investigate the mechanism of the complete reduction of O2. Formation of the Fea32+-CN- derivative contributes to the trigonal planar coordination of CuB1+ and displaces one of its three coordinated imidazole groups while a water molecule becomes hydrogen-bonded to both the CN- ligand and the hydroxyl group of Tyr244. When O2 is bound to Fea32+, it is negatively polarized (O2-), and expected to induce the same structural change induced by CN-. This allows O2- to receive three electron equivalents non-sequentially from CuB1+, Fea33+ and Tyr-OH, providing complete reduction of O2 with minimization of production of active oxygen species. The proton pumping pathway of bovine CcO comprises a hydrogen bond network and a water channel which extend to the positive and negative side surfaces, respectively. Protons transferred through the water channel are pumped through the hydrogen-bond network electrostatically with positive charge created at the Fea center by electron donation to the O2 reduction site. Binding of CO or NO to Fea32+ induces significant narrowing of a section of the water channel near the hydrogen-bond network junction, which prevents access of water molecules to the network. In a similar manner, O2 binding to Fea32+ is expected to prevent access of water molecules to the hydrogen-bond network. This blocks proton back-leak from the network and provides an efficient gate for proton pumping.


Cho, Jaeheung, Sarangi, Ritimukta, Kang, Hye Yeon, Lee, Jung Yoon, Kubo, Minoru, Ogura, Takashi, Solomon, Edward and Nam, Wonwoo.

Synthesis, Structural and Spectroscopic Characterization, and Reactivities of Mononuclear Cobalt(III)-Peroxo Complexes

J. Am. Chem. Soc., 132, 16977–16986 (2010).


Metaldioxygen adducts are key intermediates detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. In this study, mononuclear cobalt(III)peroxo complexes bearing tetraazamacrocyclic ligands, [Co(12-TMC)(O2)]+ and [Co(13-TMC)(O2)]+, were synthesized by reacting [Co(12-TMC)(CH3CN)]2+ and [Co(13-TMC)(CH3CN)]2+, respectively, with H2O2 in the presence of triethylamine. The mononuclear cobalt(III)peroxo intermediates were isolated and characterized by various spectroscopic techniques and X-ray crystallography, and the structural and spectroscopic characterization demonstrated unambiguously that the peroxo ligand is bound in a side-on ƒΕ2 fashion. The OO bond stretching frequency of [Co(12-TMC)(O2)]+ and [Co(13-TMC)(O2)]+ was determined to be 902 cm1 by resonance Raman spectroscopy. The structural properties of the CoO2 core in both complexes are nearly identical; the OO bond distances of [Co(12-TMC)(O2)]+ and [Co(13-TMC)(O2)]+ were 1.4389(17) Å and 1.438(6) Å, respectively. The cobalt(III)peroxo complexes showed reactivities in the oxidation of aldehydes and O2-transfer reactions. In the aldehyde oxidation reactions, the nucleophilic reactivity of the cobaltperoxo complexes was significantly dependent on the ring size of the macrocyclic ligands, with the reactivity of [Co(13-TMC)(O2)]+ > [Co(12-TMC)(O2)]+. In the O2-transfer reactions, the cobalt(III)peroxo complexes transferred the bound peroxo group to a manganese(II) complex, affording the corresponding cobalt(II) and manganese(III)peroxo complexes. The reactivity of the cobaltperoxo complexes in O2-transfer was also significantly dependent on the ring size of tetraazamacrocycles, and the reactivity order in the O2-transfer reactions was the same as that observed in the aldehyde oxidation reactions.


Sun Hee Kim, Hyejin Park, Mi Sook Seo, Minoru Kubo, Takashi Ogura, Jan Klajn, Daniel T. Gryko, Joan Selverstone Valentine, and Wonwoo Nam

Reversible O-O Bond Cleavage and Formation between Mn(IV)–Peroxo and Mn(V)–Oxo Corroles

J. Am. Chem. Soc., 132, 14030–14032 (2010).


Mn(IV)-peroxo and Mn(V)-oxo corroles were synthesized and characterized with various spectroscopic techniques. The intermediates were directly used in OO bond cleavage and formation reactions. Upon addition of proton to the Mn(IV)-peroxo corrole, the formation of the Mn(V)-oxo corrole was observed. Interestingly, addition of base to the Mn(V)-oxo corrole afforded the formation of the Mn(IV)-peroxo corrole. Thus, we were able to report the first example of reversible OO bond cleavage and formation reactions using in situ generated Mn(IV)-peroxo and Mn(V)-oxo corroles.


Takahiko Kojima, Yuichirou Hirai, Tomoya Ishizuka, Yoshihito Shiota, Kazunari Yoshizawa, Kenichiro Ikemura, Takashi Ogura, and Shunichi Fukuzumi

A Low-Spin Ruthenium(IV)–Oxido Complex : Does the Spin State Have an Impact on the Reactivity?

Angew. Chem. Int. Ed. 49, 8449–8453 (2010).


A ruthenium(II)–aqua complex bearing a pentadentate pyridylamine with a carboxylate group as a ligand affords a seven-coordinate low-spin (S=0) ruthenium(IV)–oxo complex (see structure) by oxidation through proton-coupled electron transfer. Comparison of the reactivity of the low-spin and an intermediate-spin (S=1) RuIV–oxo complexes revealed that the spin state does not affect the reactivity of catalytic oxidation of organic compounds.




Maeda S, Nakagawa S, Suga M, Yamashita E, Oshima A, Fujiyoshi Y, Tsukihara T.

Structure of the connexin 26 gap junction channel at 3.5 Å resolution.

Nature 458, 597-602 (2009)


Gap junctions consist of arrays of intercellular channels between adjacent cells that permit the exchange of ions and small molecules. Here we report the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 Å resolution, and discuss structural determinants of solute transport through the channel. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a positively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage.


Okada C, Yamashita E, Lee SJ, Shibata S, Katahira J, Nakagawa A, Yoneda Y, Tsukihara T.

A high-resolution structure of the pre-microRNA nuclear export machinery.

Science@326, 1275-1279 (2009)


Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3' overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt-like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3' overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.


Tanaka H, Kato K, Yamashita E, Sumizawa T, Zhou Y, Yao M, Iwasaki K, Yoshimura M, Tsukihara T.

The structure of rat liver vault at 3.5 angstrom resolution.

Science 323, 384-388 (2009)


Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.


Cho J, Sarangi R, Annaraj J, Kim SY, Kubo M, Ogura T, Solomon EI, Nam W.

Geometric and electronic structure and reactivity of a mononuclear eside-onf nickel(III)–peroxo complex.

Nature Chemistry 1, 568-572 (2009)


AbstractMetal-dioxygen adducts, such as metal-superoxo and -peroxo species, are key intermediates often detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. The synthesis and spectroscopic characterization of an end-on nickel(II)-superoxo complex with a 14-membered macrocyclic ligand was reported previously. Here we report the isolation, spectroscopic characterization, and high-resolution crystal structure of a mononuclear side-on nickel(III)-peroxo complex with a 12-membered macrocyclic ligand, [Ni(12-TMC)(O2)]+ (1) (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane). In contrast to the end-on nickel(II)-superoxo complex, the nickel(III)-peroxo complex is not reactive in electrophilic reactions, but is capable of conducting nucleophilic reactions. The nickel(III)-peroxo complex transfers the bound dioxygen to manganese(II) complexes, thus affording the corresponding nickel(II) and manganese(III)-peroxo complexes. Our results demonstrate the significance of supporting ligands in tuning the geometric and electronic structures and reactivities of metal–O2 intermediates that have been shown to have biological as well as synthetic usefulness in biomimetic reactions.


Terawaki SI, Kitano K, Mori T, Zhai Y, Higuchi Y, Itoh N, Watanabe T, Kaibuchi K, Hakoshima T.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module.

EMBO J.29, 236-250 (2009)


Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH-CC-Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH-CC-Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the beta-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.


Kawakami K, Umena Y, Kamiya N, Shen J-R

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography.

Proc Natl Acad Sci U S A.106, 8567-8572 (2009)


The chloride ion, Cl-, is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn4Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl- with a bromide ion (Br-) or an iodide ion (I-) in PSII and analyzed the crystal structures of PSII with Br- and I- substitutions. Substitution of Cl- with Br- did not inhibit oxygen evolution, whereas substitution of Cl- with I- completely inhibited oxygen evolution, indicating the efficient replacement of Cl- by I-. PSII with Br- and I- substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of theMn4Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn4Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl- anions are required to maintain the coordination structure of theMn4Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.


Aoyama H, Muramoto K, Shinzawa-Itoh K, Hirata K, Yamashita E, Tsukihara T, Ogura T, Yoshikawa S.

A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump.

Proc Natl Acad Sci U S A. 106, 2165-2169 (2009)


The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-Å resolution by limiting the X-ray dose for each shot and by using many ( approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O2 reduction site (Fe3+-O--O--Cu2+), in contrast to a ferric hydroxide (Fe3+-OH-) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O2 reduction site suppresses the proton pumping function.


Takahashi, T., Inoue-Kashino, N., Ozawa, S., Takahashi, Y., Kashino, Y., and Satoh, K.

Photosystem II complex in vivo is a monomer

J. Biol. Chem. 284, 15598-15606 (2009)


Photosystem II (PS II) complexes are membrane protein complexes that are composed of >20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is approximately 650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.


Kida, Y., Morimoto, F., and Sakaguchi, M.

Signal-anchor sequence provides motive force for polypeptide-chain translocation through the ER membrane

J. Biol. Chem. 284, 2861-2866 (2009)


Many proteins are translocated across and integrated into the endoplasmic reticulum membrane. The type I signal anchor sequence mediates the translocation of its preceding region through the endoplasmic reticulum membrane, but the source of the motive force has been unclear. Here, we characterized the motive force for N-terminal domain translocation using two probes. First, an Ig-like domain of the muscle protein titin (I27 domain) or its mutants were fused to the N termini, and translocation was examined in a cell-free translation system supplemented with rough microsomal membrane. The N-terminal translocation efficiencies correlated with the mechanical instabilities of the I27 mutants. When the I27 domain was separated from the signal anchor sequence by inserting a spacer, even the most unstable mutant stalled on the cytoplasmic side, whereas its downstream portion spanned the membrane. Proline insertion into the signal anchor sequence also caused a large translocation defect. Second, a streptavidin-binding peptide tag was fused to the N terminus. Titration of streptavidin in the translation system allowed us to estimate the translocation motive force operative on the tag. The motive force was decreased by the proline insertion into the signal anchor sequence as well as by separation from the signal anchor sequence. When the streptavidin-binding peptide tag was separated from the signal anchor, the proline insertion did not induce further deficits in the motive force for the tag. On the basis of the findings obtained by using these two independent techniques, we conclude that the signal sequence itself provides the motive force for N-terminal domain translocation within a limited upstream region.


Hayashi T, Harada K, Sakurai K, Shimada H, and Hirota S

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site

J. Am. Chem. Soc. 131, 1398-1400 (2009)


Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chains interact with Arg, His, and/or Asp residues. To understand the role of the heme-7-propionate side chain, we prepared reconstituted P450cam with an artificial one-legged heme which has a methyl group at the position of the 7-propionate. Removal of 7-propionate dramatically decreases the d-camphor affinity by 3 orders of magnitude relative to that of the wild-type enzyme, and spectroscopic data indicate that 74% of the ferric P450cam exhibits a low-spin state owing to water molecule occupancy in the substrate-binding site under the normal assay conditions. Thus, the monooxygenase activity of the reconstituted protein is remarkably low due to the decrease in the rate of the first electron transfer from reduced putidaredoxin, whereas 87% of oxidized NADH was utilized to produce 5-hydroxy-d-camphor without any significant uncoupling reactions. X-ray structural analysis of the reconstituted enzyme reveals a novel water array extending from the substrate-binding site to bulk solvent through the position occupied by 7-propionate. This water array appears without causing any major changes in the protein structure with the notable exception of conformational changes occurring at Asp297 and Gln322 residues. We propose that the 7-propionate forms a barrier against entry of bulk water molecules and therefore in combination with Asp297, Arg299, and Gln322 plays an essential role in the process of elimination of the substrate-binding site water cluster which occurs upon d-camphor binding.


Liu JG, Ohta T, Yamaguchi S, Ogura T, Sakamoto S, Maeda Y and Naruta Y

Spectroscopic Characterization of a Hydroperoxo-Heme Intermediate: Conversion of a Side-On Peroxo to an End-On Hydroperoxo Complex.

Angew. Chem. Int. Ed. 48, 9262 - 9267 (2009)


Protonation of a closed seven-coordinate side-on heme peroxide can switch its spin state from high- to low-spin and convert the η2 binding fashion into a monodentate configuration to afford the corresponding hydroperoxide. The end-on low-spin ferric hydroperoxo-heme species bear a covalently appended axial imidazole ligand and are easily prepared. This reaction represents the first example of a spin-state and binding-mode switch of heme peroxide that is triggered by protonation.



Ohtsubo M, Yasunaga S, Ohno Y, Tsumura M, Okada S, Ishikawa N, Shirao K, Kikuchi A, Nishitani H, Kobayashi M, Takihara Y.

Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.

Proc Natl Acad Sci U S A. 105, 10396-10401 (2008)


Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at

lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the

INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.


H. Mizuno, T. K. Mal, M. Welchli, A. Kikuchi, T. Fukano, R. Ando, J. Jeyakanthan, J. Taka, Y. Shiro, M. Ikura, A. Miyawaki

Light-dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein

Proc. Natl. Acad. Sci. USA 105, 9227-9232 (2008)


The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood, because the crystal structures of the bright state of the protein did not provide an answer to the mechanism of the photochromism, and structural determination of the dark state has been elusive. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. Light-induced changes in interactions between the chromophore and ƒΐ-barrel are responsible for switching between the two states. In the bright state, the apex of the chromophore tethers to the barrel by a hydrogen bond, and an imidazole ring protruding from the barrel stabilizes the plane of the chromophore. These interactions are disrupted by strong illumination with blue light, and the chromophore, together with a part of the ƒΐ-barrel, becomes flexible, leading to a nonradiative decay process.


Se-Young Son, Jichun Ma, Youhei Kondou, Masato Yoshimura, Eiki Yamashita, and Tomitake Tsukihara.

Structure of human monoamine oxidase at 2.2 Å resolution: The control of opening the entry for substrates/inhibitors.

Proc. Natl. Acad. Sci. USA 105, 5739-5744 (2008)


The mitochondrial outer membrane-anchored monoamine oxidase (MAO) is a biochemically important flavoenzyme that catalyzes the deamination of biogenic and xenobiotic amines. Its two subtypes, MAOA and MAOB, are linked to several psychiatric disorders and therefore are interesting targets for drug design. To understand the relationship between structure and function of this enzyme, we extended our previous low-resolution rat MAOA structure to the high-resolution wild-type and G110A mutant human MAOA structures at 2.2 and 2.17 Å, respectively. The high-resolution MAOA structures are similar to those of rat MAOA and human MAOB, but different from the known structure of human MAOA [De Colibus L, et al. (2005) Proc Natl Acad Sci USA 102:12684-12689], specifically regarding residues 108-118 and 210-216, which surround the substrate/inhibitor cavity. The results confirm that the inhibitor selectivity of MAOA and MAOB is caused by the structural differences arising from Ile-335 in MAOA vs. Tyr-326 in MAOB. The structures exhibit a C-terminal transmembrane helix with clear electron density, as is also seen in rat MAOA. Mutations on one residue of loop 108-118, G110, which is far from the active center but close to the membrane surface, cause the solubilized enzyme to undergo a dramatic drop in activity, but have less effect when the enzyme is anchored in the membrane. These results suggest that the flexibility of loop 108-118, facilitated by anchoring the enzyme into the membrane, is essential for controlling substrate access to the active site. We report on the observation of the structure-function relationship between a transmembrane helical anchor and an extra-membrane domain.


Nakamura-Ogiso, E., Matsuno-Yagi, A., Yoshikawa, S., Yagi, T. and Ohnishi, T.

Iron-sulfur cluster N5 is coordinated by a HXXXCXXCXXXXXC motife in the NuoG subunit of E. coli NADH:quinine oxidoreductase (Complex I)

J. Biol. Chem. (2008) 283, 25979-87.


NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)3 ligands in NuoG.


El-Mashtoly, S.F., S. Nakashima, A. Tanaka, T. Shimizu, and T. Kitagawa

Roles of Arg-97 and Tyr-113 in regulation of distal ligand binding to heme in Ec Dos protein: Resonance Raman and mutation study.

J. Biol. Chem. 283, 19000-19010 (2008)


The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O2, CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 ¨ Ile, Met-95 ¨ Ala, Arg-97 ¨ Ile, Arg-97 ¨ Ala, Arg-97 ¨ Glu, Phe-113 ¨ Leu, and Phe-113 ¨ Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.


Nishitani H, Shiomi Y, Iida H, Michishita M, Takami T, Tsurimoto T.

CDK inhibitor p21 is degraded by a PCNA coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation.

J Biol Chem. Aug 14. [Epub ahead of print] (2008)


Previous reports showed that chromatin associated PCNA couples DNA replication with Cul4-DDB1Cdt2 dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA binding protein, is also degraded both in S phase and after UV irradiation. Here, we show that p21 is degraded by the same ubiquitin -proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1Cdt2 were silenced, or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co- immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1Cdt2 complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G2 phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1Cdt2. Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.


S. Hirano, S. Asamizu, H. Onaka, Y. Shiro, S. Nagano

Crystal Structure of VioE, a Key Player in the Construction of the Molecular Skelton of Violacein

J. Biol. Chem. 283, 6459-6466 (2008)


Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate gcompound Xh, which is produced from L-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits that each forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE/substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE/phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation due to proximity and orientation effects.


El-Mashtoly, S.F., Y. Gu, H. Yoshimura, S. Yoshioka, S. Aono, and T. Kitagawa

Protein conformation changes of HemAT-Bs upon ligand binding probed by ultraviolet resonance Raman spectroscopy.

J. Biol. Chem. 283, 6942-6949 (2008)


HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O2-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr70 in the heme distal side and Tyr133 and Trp132 from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2, but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme-bound O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2 binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.


Tosha, T., N. Kagawa, M. Arase, M.R. Waterman, and T. Kitagawa

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome p450 steroid 21-hydroxylase proved by Raman spectroscopy.

J. Biol. Chem. 283, 3708-3717 (2008)


We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17ƒΏ-hydroxyprogesterone. The uncoupled H2O2 formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17ƒΏ-hydroxyprogesterone (15% of NADPH consumed), indicating that 17ƒΏ-hydroxyprogesterone can better facilitate the O–O bond scission. In relation to this, it is noted that the O–O stretching mode (ƒΛO–O) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17ƒΏ-hydroxyprogesterone-bound enzyme gave single (at 1137 cm–1) or split ƒΛO–O bands (at 1124 and 1138 cm–1), respectively, demonstrating the presence of two forms for the latter. In contrast to ƒΛO–O, no corresponding difference was observed for the Fe-O2 stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm–1) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17ƒΏ-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower ƒΛO–O frequency with higher reactivity for O–O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17ƒΏ-hydroxyprogesterone than in progesterone.


Kenichiro Ikemura, Masahiro Mukai, Hideo Shimada, Tomitake Tsukihara, Satoru Yamaguchi, Kyoko Shinzawa-Itoh, Shinya Yoshikawa and Takashi Ogura

Red-Excitation Resonance Raman Analysis of the ƒΛFe=O Mode of Ferryl-Oxo Hemoproteins.

J. Am. Chem. Soc. (2008) 130, 14384-14385.


The Raman excitation profile of the ƒΛFe=O mode of horseradish peroxidase compound II exhibits a maximum at 580 nm. This maximum is located within an absorption band with a shoulder assignable to an oxygen-to-iron charge transfer band on the longer wavelength side of the ƒΏ-band. Resonance Raman bands of the ƒΛFe=O mode of various ferryl-oxo type hemoproteins measured at 590 nm excitation indicate that many hemoproteins in the ferryl-oxo state have an oxygen-to-iron charge transfer band in the visible region. Since this red-excited resonance Raman technique causes much less photochemical damage in the proteins relative to blue-excited resonance Raman spectroscopy, it produces a higher signal-to-noise ratio and thus represents a powerful tool for investigations of ferryl-oxo intermediates of hemoproteins.


L. W. Chung, X. Li, H. Sugimoto, Y. Shiro, K. Morokuma

Density Functional Theory Study on a Missing Piece in Understanding of Heme Chemistry: The Reaction Mechanism for Indoleamine 2,3-dioxygenase and Tryptophan 2,3-dioxygenase

J. Am. Chem. Soc. 130, 12299-12309 (2008)


Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing dioxygenases and catalyze oxidative cleavage of the pyrrole ring of L-tryptophan. On the basis of three recent crystal structures of these heme-containing dioxygenases, two new mechanistic pathways were proposed by several groups. Both pathways start with electrophilic addition of the Fe(II)-bound dioxygen concerted with proton transfer (oxygen ene-type reaction), followed by either formation of a dioxetane intermediate or Criegee-type rearrangement. However, density functional theory (DFT) calculations do not support the proposed concerted oxygen ene-type and Criegee-type rearrangement pathways. On the basis of DFT calculations, we propose a new mechanism for dioxygen activation in these heme systems. The mechanism involves (a) direct electrophilic addition of the Fe(II)-bound oxygen to the C2 or C3 position of the indole in a closed-shell singlet state or (b) direct radical addition of the Fe(III)-superoxide to the C2 position of the indole in a triplet (or open-shell singlet) state. Then, a radical-recombination or nearly barrierless charge-recombination step from the resultant diradical or zwitterionic intermediates, respectively, proceeds to afford metastable dioxetane intermediates, followed by ring-opening of the dioxetanes. Alternatively, homolytic O-O bond cleavage from the diradical intermediate followed by oxo attack and facile C2-C3 bond cleavage could compete with the dioxetane formation pathway. Effects of ionization of the imidazole and negatively charged oxyporphyrin complex on the key dioxygen activation process are also studied.


Y. Wang, H. Hirao, H. Chen, H. Onaka, S. Nagano, S, Shaik

Electron Transfer Activation of Chromopyrrolic Acid by Cytochrome P450 En Route to the Formation of an Antitumor Indolocarbazol Derivative: Theory Supports Experiment

J. Am. Chem. Soc. 130, 7170-7171 (2008)


QM/MM calculations support experiment and show that StaP is a P450 that functions like a peroxidase: its active species is the one-electron-reduced Cpd II species with a radical on CPA, by analogy to cytochrome c peroxidase (CcP), and its reaction with the substrate proceeds by overall proton-coupled electron transfer (PCET), in analogy to the corresponding mechanism in horseradish peroxidase (HRP). The electron transfer is enabled by His250, the presence of carboxylate groups in CPA, and by the H-bonding network that tunes the energetic of the process. Theory supports experiment but reveals some novel aspects of this unusual P450.


Masato Kujime, Chiemi Izumi, Masaaki Tomura, Masahiko Hada, and Hiroshi Fujii

Effect of a Tridentate Ligand on the Structure, Electronic Structure, and Reactivity of the Copper(I) Nitrite Complex: Role of the Conserved Three-Histidine Ligand Environment of the Type-2 Copper Site in Copper- Containing Nitrite Reductases

J. Am. Chem. Soc. 2008, 130, 6088-6098


It is postulated that the copper(I) nitrite complex is a key reaction intermediate of copper containing nitrite reductases (Cu-NiRs), which catalyze the reduction of nitrite to nitric oxide (NO) gas in bacterial denitrification.  To investigate the structure-function relationship of Cu-NiR, we prepared five new copper(I) nitrite complexes with sterically hindered tris(4-imidazolyl)carbinols or tris(1-pyrazolyl)methanes. This study suggests that the (His)3 ligand environment is essential for acceleration of the Cu-NiR reaction. Change in the reactivity of Cu-NiR model complexes can be explained by the structural and spectroscopic characterizations and the molecular orbital calculations presented in this paper. Based on these results, the functional role of the (His)3 ligand environment in Cu-NiR is discussed.


Kunishita, A., Ishimaru, H., Nakashima, S., Ogura, T. and Itoh, S.

Reactivity of Mononuclear Alkylperoxo Copper(II) Complex. O-O Bond Cleavage and C-H Bond Activation.

J. Am. Chem. Soc. 130, 4244-4245 (2008)


A detailed reactivity study has been carried out for the first time on a new mononuclear alkylperoxo copper(II) complex, which is generated by the reaction of copper(II) complex supported by the bis(pyridylmethyl)amine tridentate ligand containing a phenyl group at the 6-position of the pyridine donor groups and cumene hydroperoxide (CmOOH) in CH3CN. The cumylperoxo copper(II) complex thus obtained has been found to undergo homolytic cleavage of the O-O bond and induce C-H bond activation of exogenous substrates, providing important insights into the catalytic mechanism of copper monooxygenases.


Harada, K., Sakurai, K., Ikemura, K., Ogura, T., Hirota, S., Shimada, H., and Hayashi, T.,

Evaluation of the Functional Role of the Heme-6-Propionate Side Chain in Cytochrome P450cam,

J. Am. Chem. Soc. 130, 432-433 (2008)


Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via non-covalent interactions. Heme-6-propionate, one of the two heme-propionate side chains, forms hydrogen bonding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional roles of the 6-propionate side chain in P450cam using a reconstituted protein with 6-depropionate-6-methylated protoheme IX (one-legged heme). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate does not significantly have an influence on the enzyme property. In contrast, its removal decreased the affinity of putidaredoxin (Pdx) by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. However, the rate of electron transfer from Pdx to P450cam was not significantly changed, which rules out the previous proposal that the 6-propionate via Arg 112 is a major electron transfer pathway. Resonance Raman experiments indicate that removal of the 6-propionate weakens the Fe–S bond strength and produces an inactive P420 species. The X-ray structure of the reconstituted protein at 1.55 Å resolution, highly superimposable with that of the wild type protein, suggests the bulk water is accessible to the Cys356 heme ligand relative to the wild type protein. Lengthening of the Fe–S bond and the water accessibility could facilitate protonation of thiolate anion to thiol, resulting in readily formation of the inactive P420 species. Therefore, the d-camphor hydroxylation reaction requires a 6-propionate-protein matrix interaction to maintain an active P450 species.




Kurotaki Y, Hatta K, Nakao K, Nabeshima Y, Fujimori T.

Blastocyst axis is specified independently of early cell lineage but aligns with the ZP shape.

Science (2007) 316, 719-723


The mechanisms controlling the establishment of the embryonic-abembryonic (E-Ab) axis of the mammalian blastocyst are controversial. We used in vitro time-lapse imaging and in vivo lineage labeling to provide evidence that the E-Ab axis of the mouse blastocyst is generated independently of early cell lineage. Rather, both the boundary between two-cell blastomeres and the E-Ab axis of the blastocyst align relative to the ellipsoidal shape of the zona pellucida (ZP), an extraembryonic structure. Lack of correlation between cell lineage and the E-Ab axis can be explained by the rotation of the embryo within the ZP.


Ogo, S., Kabe, R., Uehara, K., Kure, B., Nishimura, T., Menon, S. C., Harada, R., Fukuzumi, S., Higuchi, Y., Ohhara, T., Tamada, A. Kuroki, Y.

A dinuclear Ni(mu-H)Ru complex derived from H2.

Science (2007) 316 585-587


Models of the active site in [NiFe]hydrogenase enzymes have proven challenging to prepare. We isolated a paramagnetic dinuclear nickel-ruthenium complex with a bridging hydrido ligand from the heterolytic cleavage of H2 by a dinuclear NiRu aqua complex in water under ambient conditions (20 degrees C and 1 atmosphere pressure). The structure of the hexacoordinate Ni(mu-H)Ru complex was unequivocally determined by neutron diffraction analysis, and it comes closest to an effective analog for the core structure of the proposed active form of the enzyme.


K.Adachi., K.Oiwa., T.Nishizaka., S.Furuike., H.Noji., H. Itoh., M. Yoshida., K. Kinoshita, Jr.,
Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation.,
Cell 130, 1309-321 (2007)


F1-ATPase is a rotary molecular motor that proceeds in 120‹ steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F1 that phosphate release drives the last 40‹ of the 120‹ step, and that the 40‹ rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F1 is forced to rotate slowly, that release of Cy3-ADP occurs at 240‹ after it is bound as Cy3-ATP at 0‹. This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.


Xouri, G., Squire, A., Dimaki, M., Geverts, B., Verveer, P., Taraviras, S., Nishitani, H., Houtsmuller, A., Bastiaens, P., Lygerou, Z.

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

EMBO J. (2007) 26 1303-1314


To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.



Kyoko Shinzawa-Itoh, Hiroshi Aoyama, Kazumasa Muramoto, Hirohito Terada, Tsuyoshi Kurauchi, Yoshiki Tadehara, Akiko Yamasaki, Takashi Sugimura, Sadamu Kurono, Kazuo Tsujimoto, Tsunehiro Mizushima, Eiki Yamashita, Tomitake Tsukihara and Shinya Yoshikawa

Structures and physiological roles of thirteen integral lipids of bovine heart cytochrome c oxidase

EMBO J. (2007) 26, 1713-1725.


All 13 lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, were identified in a crystalline bovine heart cytochrome c oxidase (CcO) preparation. The chain lengths and unsaturated bond positions of the fatty acid moieties determined by mass spectrometry suggest that each lipid head group identifies its specific binding site within CcOs. The X-ray structure demonstrates that the flexibility of the fatty acid tails facilitates their effective space-filling functions and that the four phospholipids stabilize the CcO dimer. Binding of dicyclohexylcarbodiimide to the O2 transfer pathway of CcO causes two palmitate tails of phosphatidylglycerols to block the pathway, suggesting that the palmitates control the O2 transfer process. The phosphatidylglycerol with vaccenate (cis-ƒ’11-octadecenoate) was found in CcOs of bovine and Paracoccus denitrificans, the ancestor of mitochondrion, indicating that the vaccenate is conserved in bovine CcO in spite of the abundance of oleate (cis-ƒ’9-octadecenoate). The X-ray structure indicates that the protein moiety selects cis-vaccenate near the O2 transfer pathway against trans-vaccenate. These results suggest that vaccenate plays a critical role in the O2 transfer mechanism.


Xouri G, Squire A, Dimaki M, Geverts B, Verveer PJ, Taraviras S, Nishitani H, Houtsmuller AB, Bastiaens PI, Lygerou Z.

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

EMBO J. 26, 1303-1314 (2007)


To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to

chromatin provide spatio-temporal control of the licensing process.


Schwarz-Romond, T., Fiedler, M., Shibata, N., Jonathan, P., Butler, G., Kikuchi, A., Higuchi Y. and Bienz, M.

The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.

Nature Structural and Molecular Biology (2007) 14 484-492


The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of beta-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.


M. Makino, H. Sugimoto, Y. Shiro, S. Asamizu, H. Onaka, S. Nagano

Crystal Structures and Catalytic Mechanism of Cytochrome P450 StaP That Produces The Indolocarbazole Skeleton

Proc. Natl. Acad. Sci. USA 104, 11591-11596 (2007)


Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C–C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped ƒΞ-ƒΞ interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP–CPA complex, we propose that C–C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738–740]. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.


Sato, A., Y. Gao, T. Kitagawa, and Y. Mizutani

Primary protein response after ligand photodissociation in carbonmonoxy myoglobin.

Proc. Natl. Acad. Sci. U.S.A. 104A 9627-9632 2007.


Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that was completed within the instrument response time. The intensity decrease was followed by an intensity recovery with a time constant of ≈50 ps and lasted up to 1 ns. The time-resolved UVRR study of the myoglobin mutants demonstrated that the hydrophobicity of environments around Trp-14 decreased, whereas that around Trp-7 barely changed in the primary protein response. The present data indicate that displacement of the E helix toward the heme occurs within the instrument response time and that movement of the FG corner takes place with a time constant of 2 ps. The finding that the instantaneous motion of the E helix strongly suggests a mechanism in which protein structural changes are propagated from the heme to the A helix through the E helix motion.


Zhang, L., Sato, Y., Hessa, T., von Heijne, G., Lee, J.-K., Kodama, I., Sakaguchi, M., and Uozumi. N.

Contribution of hydrophobic and electrostatic interactions to the membrane integration of the Shaker K+-channel voltage-sensor domain.

Proc. Natl. Acad. Sci. USA. 104, 8263-8268 (2007)


Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels (K(v) channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant K(v) channel KAT1, the S2, S3, and S4 transmembrane helices insert cooperatively, because the S3, S4, and S3-S4 segments do not have any membrane insertion ability by themselves. Here we show that, in the Drosophila Shaker K(v) channel, which has a more hydrophobic S3 helix than KAT1, S3 can both insert into the membrane by itself and mediate the insertion of the S3-S4 segment in the absence of S2. An engineered KAT1 S3-S4 segment in which the hydrophobicity of S3 was increased or where S3 was replaced by Shaker S3 behaves as Shaker S3-S4. Electrostatic interactions among charged residues in S2, S3, and S4, including the salt bridges between E283 or E293 in S2 and R368 in S4, are required for fully efficient membrane insertion of the Shaker voltage-sensor domain. These results suggest that cooperative insertion of the voltage-sensor transmembrane helices is a property common to K(v) channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces.


Kazumasa Muramoto, Kunio Hirata, Kyoko Shinzawa-Itoh, Shinji Yoko-o, Eiki Yamashita, Hiroshi@Aoyama,Tomitake Tsukihara, and Shinya Yoshikawa

A histidine residue acting as a controlling site for dioxygen reduction and proton pumping by cytochrome c oxidase

Proc. Natl. Acad. Sci. USA (2007) 104, 7881-7886.


Cytochrome c oxidase transfers electrons and protons for dioxygen reduction coupled with proton pumping. These electron and proton transfers are tightly coupled with each other for the effective energy transduction by various unknown mechanisms. Here, we report a coupling mechanism by a histidine (His-503) at the entrance of a proton transfer pathway to the dioxygen reduction site (D-pathway) of bovine heart cytochrome c oxidase. In the reduced state, a water molecule is fixed by hydrogen bonds between His-503 and Asp-91 of the D-pathway and is linked via two water arrays extending to the molecular surface. The microenvironment of Asp-91 appears in the x-ray structure to have a proton affinity as high as that of His-503. Thus, Asp-91 and His-503 cooperatively trap, on the fixed water molecule, the proton that is transferred through the water arrays from the molecular surface. On oxidation, the His-503 imidazole plane rotates by 180 degrees to break the hydrogen bond to the protonated water and releases the proton to Asp-91. On reduction, Asp-91 donates the proton to the dioxygen reduction site through the D-pathway. The proton collection controlled by His-503 was confirmed by partial electron transfer inhibition by binding of Zn2+ and Cd2+ to His-503 in the x-ray structures. The estimated Kd for Zn2+ binding to His-503 in the x-ray structure is consistent with the reported Kd for complete proton-pumping inhibition by Zn2+ [Kannt A, Ostermann T, Muller H, Ruitenberg M (2001) FEBS Lett 503:142-146]. These results suggest that His-503 couples the proton transfer for dioxygen reduction with the proton pumping.


Kunitoshi Shimokata, Yukie Katayama, Haruka Murayama, Makoto Suematsu, Tomitake Tsukihara, Kazumasa Muramoto, Hiroshi Aoyama, Shinya Yoshikawa and Hideo Shimada

The proton pumping pathway of bovine heart cytochrome c oxidase

Proc. Natl. Acad. Sci. USA (2007) 104, 4200-4205.


X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O2 in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O2 reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O2 reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping.


T.Kumasaka, M.Yamamoto, M.Furuichi,  M.Nakasako, A.H. Teh, M.Kimura, I.Yamaguchi and T.UekiiJASRIj: Crystal structures of blasticidin S deaminase (BSD) implications for dynamic properties of catalytic zinc.

J. Biol. Chem. 282, 37103-37111 (2007).


The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of ~0.6 Å, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 Å. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate.


Hersleth, H.-P., T. Uchida, A.K. Rohr, T. Teschner, V. Schunemann, T. Kitagawa, A. Trautwein, C.H. Gorbitz, and K.K. Andersson

Crystallographic and spectroscopic studies of peroxide-derived myoglobin compound II and occurrence of protonated FeIV-O.

J. Biol. Chem. 282, 23372-23386 (2007)


High resolution crystal structures of myoglobin in the pH range 5.2–8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe–O bond length (1.86–1.90 Å) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe–O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an FeIV–O species with a single bond. The presence of FeIV is indicated by a small isomer shift of ƒΒ = 0.07 mm/s from Mössbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.


Roukos V, Iliou MS, Nishitani H, Gentzel M, Wilm M, Taraviras S, Lygerou Z.

Geminin cleavage during apoptosis by caspase-3 alters its binding ability to the SWI/SNF subunit Brahma.

J Biol Chem. 282, 9346-9357 (2007)


Geminin has been proposed to coordinate cell cycle and differentiation events through balanced interactions with the cell cycle regulator Cdt1 and with homeobox transcription factors and chromatin remodeling activities implicated in cell fate decisions. Here we show that Geminin is cleaved in primary cells and cancer cell lines induced to undergo apoptosis by a variety of stimuli. Geminin targeting is mediated by caspase-3 both in vivo and in vitro. Two sites at the carboxyl terminus of Geminin (named C1 and C2) are cleaved by the caspase, producing truncated forms of Geminin. We provide evidence that Geminin cleavage is regulated by phosphorylation. Casein kinase II alters Geminin cleavage at site C1 in vitro, whereas mutating phosphorylation competent Ser/Thr residues proximal to site C1 affects Geminin cleavage in vivo. We show that truncated Geminin produced by cleavage at C1 can promote apoptosis. In contrast, Geminin cleaved at site C2 has lost the ability to interact with Brahma (Brm), a catalytic subunit of the SWI/SNF chromatin remodeling complex, while binding efficiently to Cdt1, indicating that targeting of Geminin during apoptosis differentially affects interactions with its binding partners.


Kida, Y., Morimoto, F., and Sakaguchi, M.

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis.

J. Cell Biol. 179, 1441-1452 (2007)


During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.


Katsumasa Kamiya, Mauro Boero, Masaru Tateno, Kenji Shiraishi, and Atsushi Oshiyama

Possible Mechanism of Proton Transfer through Peptide Groups in the H-Pathway of the Bovine Cytochrome c Oxidase.

J. Am. Chem. Soc. 129, 9663-9673 (2007)


The peptide group connecting Tyr440 and Ser441 of the bovine cytochrome coxidase is involved in a recently proposed proton-transfer path (H-path) where, at variance with other pathways (D- and K-paths), a usual hydrogen-bond network is interrupted, thus making this proton propagation rather unconventional. Our density-functional based molecular dynamics simulations show that, despite this anomaly and provided that a proton can reach a nearby water, a multistep proton-transfer pathway can become a viable pathway for such a reaction: A proton is initially transferred to the carbonyl oxygen of a keto form of the Tyr440-Ser441 peptide group [-CO-NH-], producing an imidic acid [-C(OH)-NH-] as a metastable state; the amide proton of the imidic acid is then transferred, spontaneously to the deprotonated carboxyl group of the Asp51 side chain, leading to the formation of an enol form [-C(OH)dN-] of the Tyr440-Ser441 peptide group. Then a subsequent enol-to-keto tautomerization occurs via a double proton-transfer path realized in the two adjacent Tyr440-Ser441 and Ser441-Asp442 peptide groups. An analysis of this multistep proton-transfer pathway shows that each elementary process occurs through the shortest distance, no permanent conformational changes are induced, thus preserving the X-ray crystal structure, and the reaction path is characterized by a reasonable activation barrier.


El-Mashtoly, S.F., H. Takahashi, T. Shimizu, and T. Kitagawa

Ultraviolet resonance Raman evidence for utilization of the heme 6-propionate hydrogen-bond network in signal transmission from heme to protein in Ec Dos protein.

J. Am. Chem. Soc. 129, 3556-3563 (2007)


The direct oxygen sensor protein from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of either O2 or CO molecule to a reduced heme enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report ultraviolet resonance Raman (UVRR) spectroscopic investigations of the reduced, O2- and CO-bound forms of heme-bound PAS domain of Ec DOS. The UVRR results show that heme discriminates different ligands, resulting in altered conformations in the protein moiety. Specifically, the environment around Trp53 that contacts the 2-vinyl group of heme, is changed to a more hydrophobic environment by O2 binding, whereas it is changed to a more hydrophilic environment by CO-binding. In addition, the PDE activity of the O2- and CO-bound forms for the Trp53Phe mutant is significantly decreased compared with that of the wild type (WT), demonstrating the importance of Trp53 for the catalytic reaction. On the other hand, the binding of O2 or CO to the heme produces drastic changes in the Tyr126 of Iƒΐ-strand at the surface of the sensor domain. Furthermore, we found that Asn84 forms a hydrogen bond with Tyr126 either in the O2- or CO-bound forms but not in the reduced form. Finally, the PDE activities of the ligand-bound forms for Asn84Val and Tyr126Phe mutants are significantly reduced as compared with that of WT, suggesting the importance of the hydrogen-bonding network from heme 6-propionate to Tyr126 through Asn84 in signal transmission.


Harada, K., Sakurai, K., Ikemura, K., Ogura, T., Hirota, S., Shimada, H. and Hayashi, T.

Evaluation of the Functional Role of the Heme-6-propionate Side Chain in Cytochrome P450cam.

J. Am. Chem. Soc. 130, 432-433 (2007)


Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovalent interactions. Heme-6-propionate, one of the two heme-propionate side chains, forms hydrogen-bonding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional roles of the 6-propionate side chain in P450cam using a reconstituted protein with 6-depropionate-6-methylated protoheme IX (one-legged heme). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate has no clear influence on the enzyme property. The rate of electron transfer from putidaredoxin (Pdx), a natural redox partner, to P450cam was not significantly changed, whereas, the removal of the 6-propionate decreased the affinity of Pdx by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. Resonance Raman experiments indicate that removal of the 6-propionate weakens the Fe-S bond strength. The X-ray structure of the reconstituted protein at 1.55 Å resolution is highly superimposable with that of the wild-type protein, whereas the thiolate of the Cys357 heme ligand in the reconstituted protein is visible from the protein surface owing to the lack of the 6-propionate. Lengthening of the Fe-S bond and the water accessibility could facilitate protonation of thiolate anion to thiol, explaining the observed formation of the inactive P420 species under the mild conditions. Therefore, the d-camphor hydroxylation reaction requires a 6-propionate-protein matrix interaction to maintain an active P450 species.

T. Ishikawa, H. Sakakibara, K. Oiwa
The architecture of outer dynein arms in situ.
J. Mol. Biol 368, 1249-1258 (2007)

Outer dynein arms, the force generators for axonemal motion, form arrays on microtubule doublets in situ, although they are bouquet-like complexes with separated heads of multiple heavy chains when isolated in vitro. To understand how the three heavy chains are folded in the array, we reconstructed the detailed 3D structure of outer dynein arms of Chlamydomonas flagella in situ by electron cryo-tomography and single-particle averaging. The outer dynein arm binds to the A-microtubule through three interfaces on two adjacent protofilaments, two of which probably represent the docking complex. The three AAA rings of heavy chains, seen as stacked plates, are connected in a striking manner on microtubule doublets. The tail of the ƒΏ-heavy chain, identified by analyzing the oda11 mutant, which lacks ƒΏ-heavy chain, extends from the AAA ring tilted toward the tip of the axoneme and towards the inside of the axoneme at 50‹, suggesting a three-dimensional power stroke. The neighboring outer dynein arms are connected through two filamentous structures: one at the exterior of the axoneme and the other through the ƒΏ-tail. Although the ƒΐ-tail seems to merge with the ƒΏ-tail at the internal side of the axoneme, the ƒΑ-tail is likely to extend at the exterior of the axoneme and join the AAA ring. This suggests that the fold and function of ƒΑ-heavy chain are different from those of ƒΏ and ƒΐ-chains.

H.Iwamoto., K.Oiwa., M.Kovacs., J.R.Sellers., T.Suzuki., J.Wakayama ., T.Tamura., N.Yagi.,
Diversity of structural behavior in vertebrate conventional myosins complexed with actin:
J. Mol. Biol. 369, 249-264 (2007)

Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto–S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto–S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the gconventionalh myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.


Yamaguchi, T., Omatsu, N., Morimoto, E., Nakashima, H., Ueno, K., Tanaka, T., Satouchi, K., Hirose, F., and Osumi, T.: CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation.

J. Lipid Res. 48, 1078-1089 (2007)


A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.




2) Articles in Academic Journals or Newspaper


ET. Sugimura and K. Hagiya

Di-2-methoxyethyl Azodicarboxylate (DMEAD): An Inexpensive and Separation-friendly Alternative Reagent for the Mitsunobu Reaction.

Chem. Lett. 36, 566 (2007)


EKida, Y., Morimoto, F., and Sakaguchi, M.

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis.

J. Cell Biol. 179, 1441-1452 (2007)




3) Graduate Student Poster Award at International Conference


EH. Adachi, I. Enami, T. Henmi, N. Kamiya and J.-R. Shen

Purification and crystallization of photosystem II dimer complex from a red alga Cyanidium caldarium

Outstanding Poster Award, 14th International Congress on Photosynthesis, Glasgow, England. July 22-27, 2007




4) Keynote and Invited Lectures in International Conference


Keynote Lecture

EYoshikawa, S.: Reaction mechanism of bovine heart cytochrome c oxidase, 53rd National Meeting of Italian Society of Biochemistry and Molecular Biology (Riccione, Italy, 2008, September 23-26)


Invited Lectures

EShen J.-R.@Structure and function of oxygen-evolving photosystem II., Pre-RCE Workshop on Catalysis for Efficient and Sustainable Energy, May 28, 2009, Singapore.


ETakashi OguraFThe 4th Asian Biological Inorganic Chemistry Conference, November 10-13, 2008, Jeju, Korea


EShen J.-R., Roles of chloride and small subunits in PSII function and assembly studied by X-ray crystal structural analysis, Japan-Finland Joint Seminar on Regulation and Dynamics of Photosynthesis (Helsinki), Oct. 28, 2008-Nov. 1, 2008.


ET. Matsui, Y. Shigeta and K. Hirao:Global and local structure of cis-platin and DNA base pair complex: a theoretical study, 2nd World Conference on (Ehrlich II), Oct. 2008, Nürnberg, Germany


EY. Shigeta: Quantum dynamics in terms of cumulant, Theory and Application in Computational Chemistry 2008, Shanghai Sep. 2008, China


EK. Kamiya, Y. Shigeta, A. Oshiyama: A novel proton transfer through peptide group in protein, Theory and Application in Computational Chemistry 2008, Shanghai Sep. 2008, China


EYoshikawa, S.:@Evidence for h channel proton pump in bovine cytochrome c oxidase, 15th European Bioenergetics Conference (Dublin, Ireland, 2008, July 19-24)


EShiro, Y., Nagano, S., Matsumoto, Y.FFungal and Bacterial Nitric Oxide Reductases: Their Structures and Reaction Mechanisms

Fifth International Conference on Porphyrins and Phthalocyanines, Moscow, Russia, July (2008)


EY. Shigeta:Quantum mechanics in terms of cumulant, 13th Quantum Systems in Chemistry and Physics, Lansing July 2008, USA


EH. Fujii: Role of Highly Conserved Three-Histidines Ligand Environment of Type-2 Cu Site in Cu Nitrite Reductases, 1st CMD INTERNATIONAL SYMPOSIUM - Chemical Computations Seoul, May 30 - June 1, 2008


EYoshikawa, S.FMechanism of the mitochondrial respiration, Global COE program, International Symposium on Picobiology (Kamigohri, Hyogo, Japan, 2008, March 18-19)


EYoshikawa, S.FLipid-Protein Interactions in Cytochrome c OxidaseAKeystone Symposia, "Molecular Basis for Biological Membrane Organization"(invited presentation) (Big Sky, Montana USAA2008, January 12-18j


EYoshikawa, S.FProton Pumping Mechanism of Bovine Heart Cytochrome c Oxidase, Joint Meeting of the Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress (invited presentation) (Long Beach, California, USA, 2008, February 2-6)


EK. Oiwa:Fluorescence Based Single Molecule Imaging and Nanometry on Axonemal Dyneins
7th International Weber Symposium 2008(
Kauai, Hawai 2008)


EK. Oiwa:Development of bio-nano-devices based on protein motors'function
The 5th International Symposium on Organic Molecular Electronics (
Himeji, 2008)


ETakashi SugimuraFHistory of Heterogeneous Asymmetric Catalysis

International Symposium on Catalysis and Fine Chemicals@i2007 December 16-22, Singaporej


EShen J.-R.:Functional implications from the structural studies of Photosystem II oxygen-evolving complex,

Nagoya International Symposium on Science of Molecular Assembly and Biomolecular Systems, Nagoya, Nov. 30-Dec. 1, 2007.


EMuramoto, K.F A histidine residue acting as a controlling site for dioxygen and proton pumping by cytochrome c oxidase, Workshop on the proton-pumping mechanism of mitochondorial respiratory system (Kamigohri, Hyogo, Japan, 2007, November 13)


EShiro, Y.FInter-domain and Inter-molecular Signaling Pathway in Two Component System: Histidine Kinase and Response Regulator

9th International Conference on Biology and Synchrotron Radiation, Manchester, UK, August (2007)


EM.YamamotoFProtein Crystallography Beamlines at SPring-8

International Conference in Structural Biology, (Hong Kong, China, 2007)


EK. Oiwa:Force-generation mechanism of dynein of inspired by the structure of isolated and in situ dyneins of Chlamydomonas axonemesD
Alpbach Workshop on Moleculer Motors (Alpbach, 2007j

EK. Oiwa:Force-generating mechanism of axonemal dyneins studied by cryoelectron tomography and x-ray fiber diffraction analysis.
Japan Partnership Awards Dynein Workshop (Bristol, 2007

ES. Toba, C. Mellor, J. Molloy, K. Oiwa:Single molecule imaging of axonemal dynein-fD
The Third Workshop of the UK-Japan Bionanotechnology Collaboration. (Oxford, 2007j

EK. Oiwa:The structure and mechanism of protein motors and their potential applications in the development of bio-nano-devices
D.B. Robinson Distinguished Speaker Series,
University of Alberta (Edmonton, 2007j

EK. OiwaFMolecular-scale communication inspired by protein motorsf functionsDInternational Symposium on Nano-Medicine 2007 (Okazaki, 2007j

EK. Oiwa:Dynein and its applications to nanotechnology
The 3rd Workshop of the UK-Japan Nanobiotechnology Collaboration (
Oxford, 2007j