Infrared-Detectable Group Senses Changes in Charge Density on the Nickel Center in Hydrogenase from Chromatium vinosum (original) (raw)
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Biochemistry, 2000
An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI(r), and the carbon monoxide complex, SI-CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI(u); distorted four-coordinate sites in SI(r) and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni-O bond accounts for the change in coordination number from five to four that accompanies formation of SI(r). A shortening of the Ni-Fe distance from 2.85(5) A in form B to 2.60(5) A also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI-CO complex reveals the presence of Ni-CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H(2) or H(-) was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structural differences between the Ni sites were observed.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996
Upon reduction under hydrogen-argon atmosphere, the nickel-hydrogenases generally show a characteristic rhombic EPR spectrum which is known as Ni-C. Illumination of this state at temperatures below 60 K has previously been shown to cause the disappearance of the Ni-C signal and the simultaneous appearance of two overlapping signals, here referred to as Ni-L1 and Ni-L2, which revert to the Ni-C state at higher temperatures. These phenomena have been compared in three nickel-containing hydrogenases, the [NiFe]-hydrogenases from Desulfot'ibrio gigas and Desulfovibrio fructosovorans, and the selenium-containing soluble [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. Significant differences were observed between these enzymes. (1) The Ni-C, Ni-L1 and Ni-L2 EPR spectra were almost identical for D. gigas and D. fructosovorans hydrogenases, but the rates of photoconversion of Ni-C to Ni-L1 were different, being about 5 times slower for D. fructosovorans than for D. gigas in H20. The kinetic isotope effect in 2H20/HzO was a factor of thirty in D. gigas, but only two in D. fructosovorans. Dm. baculatum hydrogenase showed almost no kinetic isotope effect on the Ni-C to Ni-L1 conversion, but an effect on the conversion of Ni-L1 to Ni-L2. The kinetic isotope effects indicate that the processes involve the movement of a hydrogen nucleus. (3) The Ni-L1 species converted to into Ni-L2 in the dark at a rate that was virtually temperature-independent below 30 K, indicative of a proton tunnelling process. (4) The conversion of Ni-L1 to Ni-L2 was partly reversed by light in Dm. baculatum hydrogenase, but not in the [NiFe]-hydrogenases. (5) Prolonged illumination of the three enzymes induced the appearance of a third light-induced signal, Ni-L3. The new signal was rhombic, with features at g = 2.41, 2.16 (the third component being unresolved) in the [NiFe]-hydrogenases and g = 2.48, 2.16, 2.03 for the [NiFeSe]-enzyme. (6) Splittings caused by by spin-spin interactions with [4Fe-4S] clusters were detected for all the illuminated signals, Ni-LI, Ni-L2 and Ni-L3. These were quantitatively different for the three enzymes. (7) Broadening of the Ni-C signals in H20 compared with 2H20 was observed in the gj and g2 components of D. gigas and D. fructosovorans hydrogenases, but not for Din. baculatum. This broadening effect was not seen with any of the Ni-L species. These comparative effects are discussed in terms of subtle differences in the structure and protein environment of the nickel site, and access to exchangeable hydrons.
JBIC Journal of Biological Inorganic Chemistry, 2004
The membrane-bound [NiFe]-hydrogenase from Allochromatium vinosum can occur in several inactive or active states. This study presents the first systematic infrared characterisation of the A. vinosum enzyme, with emphasis on the spectro-electrochemical properties of the inactive/active transition. This transition involves an energy barrier, which can be overcome at elevated temperatures. The reduced Ready enzyme can exist in two different inactive states, which are in an apparent acid-base equilibrium. It is proposed that a hydroxyl ligand in a bridging position in the Ni-Fe site is protonated and that the formed water molecule is subsequently removed. This enables the active site to bind hydrogen in a bridging position, allowing the formation of the fully active state of the enzyme. It is further shown that the active site in enzyme reduced by 1 bar H 2 can occur in three different electron paramagnetic resonance (EPR)-silent states with a different degree of protonation.
Biochemistry, 2003
For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RH ox ) and presence of hydrogen (RH +H2 ). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH +H2 state. XANES and EXAFS spectra indicate a coordination of the Ni in the RH ox and RH +H2 that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H 2 are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH ox ) to 8341.1 eV (RH +H2 ) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H 2 . One likely interpretation of the data is the detachment of one sulfur ligand in RH +H2 and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated Ni II (O,N) 2 S 3 for RH ox and six-coordinated Ni (III) -(O,N) 3 X 1 S 2 [X being either an (O,N) or H ligand] for RH +H2 . Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed. † We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft within SFB 498 (for M.H., P.L., and H.D. within projects C8 and C6 and for A.P., T.B., and B.F. within project C1). A.P., T.B., and B.F. also thank the Fonds der Chemischen Industrie for financial support. W.M.-K. is the EMBL station scientist at beamline D2 of HASYLab/DESY Hamburg.
Journal of The American Chemical Society, 2000
L-edge X-ray absorption spectroscopy has been used to study, under a variety of conditions, the electronic structure of Ni in the Ni-Fe hydrogenases from DesulfoVibrio gigas, DesulfoVibrio baculatus, and Pyrococcus furiosus. The status of the enzyme films used for these measurements was monitored by FT-IR spectroscopy. The L-edge spectra were interpreted by ligand field multiplet simulations and by comparison with data for Ni model complexes. The spectrum for Ni in D. gigas enzyme "form A" is consistent with a covalent Ni(III) species. In contrast, all of the reduced enzyme samples exhibited high spin Ni(II) spectra. The significance of the Ni(II) spin state for the structure of the hydrogenase active site is discussed. Frey, M.; Garcin, E.; Hatchikian, C.; Montet, Y.; Piras, C.; Vernede, X.; Volbeda, A.
Eur J Biochem, 1989
Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocupsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 'H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 'H and I4N were also observed by ESEEM. The nature of the interacting nuclei is discussed.
European Journal of Biochemistry, 1989
Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocupsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 'H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 'H and I4N were also observed by ESEEM. The nature of the interacting nuclei is discussed.
Biochemistry, 1999
The nickel-iron hydrogenase from Chromatium Vinosum adsorbs at a pyrolytic graphite edgeplane (PGE) electrode and catalyzes rapid interconversion of H + (aq) and H 2 at potentials expected for the half-cell reaction 2H + a H 2 , i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H 2 is extremely rapid; at 10% partial pressure H 2 , mass transport control persists even at the highest electrode rotation rates. The turnover number for H 2 oxidation lies in the range of 1500-9000 s-1 at 30°C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at-30 mV vs SHE (pH 7.0, 30°C), is assigned to the [3Fe-4S] +/0 cluster on the basis of potentiometric measurements. The second, at-301 mV and having a 1.5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S] 2+/+ clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in COinhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H 2 oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S] +/0 cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.
JBIC Journal of Biological Inorganic Chemistry, 2009
The [NiFe] hydrogenase from the sulphatereducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SI r , provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SI a , Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SI r was found to be light-sensitive (T B 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI 1961 ). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SI r in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SI r photoconversion was examined in the range between 410 and 680 nm. Lightinduced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni 2? ). In addition, at T B 40 K the CNstretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogenbonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed.