Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.12.7.2 (hydrogenase)
 3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The selenium-containing F420-nonreducing hydrogenase from Methanococcus voltae was prepared in the Nia(I) middle dotCO state. The effect of illumination on this light-sensitive species was studied. EPR studies were carried out with enzyme containing natural selenium or with enzyme enriched in 77Se. Samples were prepared with either CO or 13CO. In the Nia(I) middle dotCO state, the nuclear spins of both 77Se (I = 1/2) and 13C (I = 1/2) interacted with the nickel-based unpaired electron, suggesting that they are positioned on opposite sites of the nickel ion. In the light-induced signal, the interaction with 13CO was lost. The 77Se nuclear spin introduced an anisotropic hyperfine splitting in both the dark and light-induced EPR signals. The data on the active enzyme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.
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PMID:Interactions of 77Se and 13CO with nickel in the active site of active F420-nonreducing hydrogenase from Methanococcus voltae. 879 8

Results are presented of the first rapid-mixing/rapid-freezing studies with a [NiFe]-hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia-SR state) to the Nia-C* state was completed in less than 8 ms, a rate consistent with the H2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia-SR state did not react with CO. It was discovered, contrary to expectations, that the Nia-C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia-C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia-C* state decayed within 40 ms, due to the generation of the Nia-S.CO state (the EPR-silent state of the enzyme with bound CO). The Nia-C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Nia*.CO) by illumination at 30 K (evoking the Nia-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia-L* and the Nia*.CO states is formally monovalent.
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PMID:Pre-steady-state kinetics of the reactions of [NiFe]-hydrogenase from Chromatium vinosum with H2 and CO. 1009 43

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.
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PMID:Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16. 1930 63

The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.
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PMID:Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase. 2579 Jan 78

We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.
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PMID:Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State. 2620 14

Pulsed ENDOR and HYSCORE measurements were carried out to characterize the active site of the oxygen-tolerant NAD(+)-reducing hydrogenase of Ralstonia eutropha. The catalytically active Nia-C state exhibits a bridging hydride between iron and nickel in the active site, which is photodissociated upon illumination. Its hyperfine coupling is comparable to that of standard hydrogenases. In addition, a histidine residue could be identified, which shows hyperfine and nuclear quadrupole parameters in significant variance from comparable histidine residues that are conserved in standard [NiFe] hydrogenases, and might be related to the O2 tolerance of the enzyme.
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PMID:Active Site of the NAD(+)-Reducing Hydrogenase from Ralstonia eutropha Studied by EPR Spectroscopy. 2621 95

Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.
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PMID:Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase. 2695 69