樋口 芳樹

[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen‐tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Nia‐C and Nia‐SR’’, were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron‐sulfur clusters of the enzyme. In addition, IR and EPR experiments performed under illumination revealed an unusual photochemical response of the enzyme. Nia‐SR’’, a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Nia‐L. In contrast to the well‐known photolysis of the more oxidized hydride intermediate Nia‐C, photoconversion of Nia‐SR’’ into Nia‐L is an active‐site redox reaction that involves light‐driven electron transfer towards the enzyme’s iron‐sulfur clusters. Omitting the ground‐state intermediate Nia‐C, this direct interconversion of these two states represents a potential photochemical shortcut of the catalytic cycle that integrates multiple redox sites of the enzyme. In total, our findings reveal the non‐local redistribution of electrons via thermal and photochemical reaction channels and the potential of accelerating or controlling [NiFe] hydrogenases by light.

Geometric and electronic structure changes in the copper (Cu) centers in bilirubin oxidase (BOD) upon a four-electron reduction were investigated by quantum mechanics/molecular mechanics (QM/MM) calculations. For the QM region, the unrestricted density functional theory (UDFT) method was adopted for the open-shell system. We found new candidates of the native intermediate (NI, intermediate II) and the resting oxidized (RO) states, i.e., NI and RO₀. Elongations of the Cu-Cu atomic distances for the trinuclear Cu center (TNC) and very small structural changes around the type I Cu (T1Cu) were calculated as the results of a four-electron reduction. The QM/MM optimized structures are in good agreement with recent high-resolution X-ray structures. As the structural change in the TNC upon reduction was revealed to be the change in the size of the triangle spanned by the three Cu atoms of TNC, we introduced a new index () to characterize the specific structural change. Not only the wild-type, but also the M467Q, which mutates the amino acid residue coordinating T1Cu, were precisely analyzed in terms of their molecular orbital levels, and the optimized redox potential of T1Cu was theoreti

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/β- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.

Hydrogenase is a key enzyme for a coming hydrogen energy society, because it has strong catalytic activities on both uptake and production of dihydrogen. We, however, have to overcome the sensitivity against O2 of the enzyme, because hydrogenase is, generally, easily inactivated in the presence of O2. In this study, we have revisited the crystal structures of [NiFe]‑hydrogenase from sulfate-reducing bacterium in the several oxidized and reduced conditions. Our results revealed that the Ni‐Fe active site of the enzyme exposed into O2 showed two forms, Form-1 and Form-2. The Ni‐Fe active site in Form-1 showed the typical Ni‐B (inactive ready) structure, whereas those in Form-2 lost Ni with no relation to an exposure time to O2, and two cysteinyl sulfur ligands made a disulfide bond. On the other hand, the formation of sulfenylation of the cysteinyl ligand to Ni, which is often observed in the oxidized form, did not correlate with the Ni-elimination, but with exposure time to O2.

NAD(+) (oxidized form of NAD: nicotinamide adenine dinucleotide)-reducing soluble [ NiFe]hydrogenase (SH) is phylogenetically related to NADH (reduced form of NAD(+)): quinone oxidoreductase (complex I), but the geometrical arrangements of the subunits and Fe-S clusters are unclear. Here, we describe the crystal structures of SH in the oxidized and reduced states. The cluster arrangement is similar to that of complex I, but the subunits orientation is not, which supports the hypothesis that subunits evolved as prebuilt modules. The oxidized active site includes a six-coordinate Ni, which is unprecedented for hydrogenases, whose coordination geometry would prevent O-2 from approaching. In the reduced state showing the normal active site structure without a physiological electron acceptor, the flavin mononucleotide cofactor is dissociated, which may be caused by the oxidation state change of nearby Fe-S clusters and may suppress production of reactive oxygen species.

Previously, the Ni-SIr state of [NiFe] hydrogenase was found to convert to the Ni-SIa state by light irradiation. Herein, large activation energies and a large kinetic isotope effect were obtained for the reconversion of the Ni-SIa state to the Ni-SIr state after the Ni-SIr-to-Ni-SIa photoactivation, suggesting that the Ni-SIa state reacts with H2O and leaves a bridging hydroxo ligand for the Ni-SIr state.

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O-2 stretching band was observed at 580cm(-1) for the reduced/oxygenated heterodimer (at 554cm(-1) under an O-18(2) atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Electrostatic interactions between proteins are key factors that govern the association and reaction rate. We spectroscopically determine the second-order reaction rate constant (k) of electron transfer from [NiFe] hydrogenase (H(2)ase) to cytochrome (cyt) c(3) at various ionic strengths (I). The k value decreases with I. To analyze the results, we develop a semi-analytical formula for I dependence of k based on the assumptions that molecules are spherical and the reaction proceeds via a transition state. Fitting of the formula to the experimental data reveals that the interaction occurs in limited regions with opposite charges and with radii much smaller than those estimated from crystal structures. This suggests that local charges in H(2)ase and cyt c(3) play important roles in the reaction. Although the crystallographic data indicate a positive electrostatic potential over almost the entire surface of the proteins, there exists a small region with negative potential on H(2)ase at which the electron transfer from H(2)ase to cyt c(3) may occur. This local negative potential region is identical to the hypothetical interaction sphere predicted by the analysis. Furthermore, I dependence of k is predicted by the Adaptive Poisson-Boltzmann Solver considering all charges of the amino acids in the proteins and the configuration of H(2)ase/cyt c(3) complex. The calculation reproduces the experimental results except at extremely low I. These results indicate that the stabilization derived from the local electrostatic interaction in the H(2)ase/cyt c(3) complex overcomes the destabilization derived from the electrostatic repulsion of the overall positive charge of both proteins. (C) 2017 Elsevier B.V. All rights reserved.

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices A and B between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.

A membraneless direct electron transfer (DET)-type dihydrogen (H-2)/air-breathing biofuel cell without any mediator was constructed wherein bilirubin oxidase from Myrothecium verrucaria (BOD) and membrane-bound [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (MBH) were used as biocatalysts for the cathode and the anode, respectively, and Ketjen black-modified water proof carbon paper (KB/WPCC) was used as an electrode material. The KB/WPCC surface was modified with 2-aminobenzoic acid and p-phenylenediamine, respectively, to face the positively charged electron accepting site of BOD and the negatively charged electron-donating site of MBH to the electrode surface. A gas-diffusion system was employed for the electrodes to realize high-speed substrate supply. As result, great improvement in the current density of O2 reduction with BOD and H-2 reduction with MBH were realized at negatively and postively charged surfaces, respectively. Gas diffusion system also suppressed the oxidative inactivation of MBH at high electrode potentials. Finally, based on the improved bioanode and biocathode, a dual gas-diffusion membrane- and mediatorless H-2/air-breathing biofuel cell was constructed. The maximum power density reached 6.1 mW cm(-2) (at 0.72 V), and the open circuit voltage was 1.12 V using 1 atm of H-2 gas as a fuel at room temperature and under passive and quiescent conditions. (C) 2016 Elsevier B.V. All rights reserved.

Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of [NiFe] hydrogenases comprise a dinuclear Ni-Fe center carrying CO and CN- ligands. The catalytic activity of the standard (O-2-sensitive) [NiFe] hydrogenases vanishes under aerobic conditions. The O-2-tolerant [NiFe] hydrogenases can sustain H-2 oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented [4Fe-3S]-6Cys cluster can supply two electrons, which lead to rapid recovery of the O-2 inactivation, to the [NiFe] active site.

Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, from Escherichia coli is involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space group P2(1), with unit-cell parameters a = 49.51, b = 88.79, c = 53.95 angstrom, beta = 94.24 degrees, and deuterated CueO crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 49.91, b = 106.92, c = 262.89 angstrom. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV-Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms in the active site to those of previously reported CueO structures. These results indicate that the CueO protein prepared using the new construct is suitable for further neutron diffraction studies.

The enzyme 6-aminohexanoate-dimer hydrolase catalyzes amide synthesis. The yield of this reverse reaction in 90% t-butyl alcohol was found to vary drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft were used. Movement of the loop region and the flip-flop of Tyr170 generate a local hydrophobic environment at the catalytic center of the enzyme. Here, we propose that the shift of the internal equilibrium between the enzyme-substrate complex and enzyme-product complex by the water-excluding effect' alters the rate of the forward and reverse reactions. Moreover, we suggest that the local hydrophobic environment potentially provides a reaction center suitable for efficient amide synthesis.DatabasePDB code : Hyb-24DNY-S-187 PDB code : Hyb-24DNY-A(187) PDB code : Hyb-24DNY-G(187) PDB code : Hyb-24DN-A(112)/Ahx complex PDB code : Hyb-24DNY-A(112)/Ahx complex PDB code : Hyb-24DNY-S(187)A(112)/Ahx complex PDB code : Hyb-24DNY-A(187)A(112)/Ahx complex PDB code : Hyb-24DNY-G(187)A(112)/Ahx complex

The Ni-SIr state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F was photoactivated to its Ni-SIa state by Ar+ laser irradiation at 514.5 nm, whereas the Ni-SL state was light induced from a newly identified state, which was less active than any other identified state and existed in the "as-isolated" enzyme.

The acetate-bound form of the type II copper was found in the X-ray structure of the multicopper oxidase CueO crystallized in acetate buffer in addition to the conventional OH--bound form as the major resting form. The acetate ion was retained bound to the type II copper even after prolonged exposure of a CueO crystal to X-ray radiation, which led to the stepwise reduction of the Cu centres. However, in this study, when CueO was crystallized in citrate buffer the OH--bound form was present exclusively. This fact shows that an exogenous acetate ion reaches the type II Cu centre through the water channel constructed between domains 1 and 3 in the CueO molecule. It was also found that the enzymatic activity of CueO is enhanced in the presence of acetate ions in the solvent water.

H-2/O-2 biofuel cells utilizing hydrogenases and multicopper oxidases as bioelectrocatalysts are clean, sustainable, and environmentally friendly power devices. In this study, we constructed a novel gas diffusion bioelectrode with a sheet of waterproof carbon cloth as the electrode base and optimized the hydrophilicity/hydrophobicity of the electrode for both high gas permeability and high direct electron transfer bioelectrocatalytic activity. The electrode exhibited a large current density of about 10 mA cm(-2) in the steady-state for both H-2 oxidation and O-2 reduction. The biocathode and the bioanode were coupled to construct a gas diffusion H-2/O-2 biofuel cell. The dual gas diffusion system allowed the separate supply of gaseous substrates (H-2 and O-2) to the bioanode and biocathode, with consequent suppression of the oxidative inhibition of the hydrogenases. The cell exhibited a maximum power density of 8.4 mW cm(-2) at a cell voltage of 0.7 V under quiescent conditions.