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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 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.
J Biol Inorg Chem 2004 Sep
PMID:The activation of the [NiFe]-hydrogenase from Allochromatium vinosum. An infrared spectro-electrochemical study. 1524 88

Plant geranylgeranyl hydrogenase (CHL P) reduces free geranylgeranyl diphosphate to phytil diphosphate, which provides the side chain to chlorophylls, tocopherols, and plastoquinones. In peach, the single copy gene (PpCHL P) encodes a deduced product of 51.68 kDa, which harbours a transit peptide for cytoplasm-to-chloroplast transport and a nicotinamide binding domain. The PpCHL P message was abundant in chlorophyll-containing tissues and flower organs, but barely detected in the roots and mesocarp of ripening fruits, suggesting that transcription was related to plastid types and maturation. The message was not revealed in shoot apical meristems, but spread thoroughly in leaf cells during the early stages and was located mainly in the palisade of mature leaves, which exhibited higher transcript levels than young ones. Hence, the transcription of PpCHL P was likely to be regulated during leaf development. Gene expression was monitored in leaves responding to natural dark, cold, wounding, stress by imposed darkening, and during the curl disease. Transcription was stimulated by light, but repressed by dark and cold stress. In darkened leaves, the PpCHL P message was augmented concomitantly with that of CATALASE. In wounded leaves, the message decreased, but recovered rapidly, whereas in curled leaves, a reduction in gene expression was related to leaf damage intensity. However, transcript signals increased locally both in cells mechanically wounded by a needle and in those naturally injured by the pathogenic fungus Taphrina deformans. These data suggest that PpCHL P expression was regulated by photosynthetic activity and was possibly involved in the defence response.
J Exp Bot 2004 Sep
PMID:The gene geranylgeranyl reductase of peach (Prunus persica [L.] Batsch) is regulated during leaf development and responds differentially to distinct stress factors. 1528 45

The active centers of NiFe hydrogenase from Desulfovibrio vulgaris Miyazaki F have been located in the electron-density map calculated at 4 A resolution. The electron-density map based on five heavy-atom derivatives showed four strong peaks which were clearly distinguished from the protein region. These strong densities have been successfully assigned to three iron-sulfur clusters and one Ni atom by a difference Fourier technique with coefficients of the best phases from the multiple isomorphous replacement (MIR) method and structure factors obtained at five wavelengths (1.040, 1.487, 1.730, 1.743 and 1.750 A) with the use of a synchrotron radiation source. Four active centers are approximately lined up at a distance of ca 13 A, which seems reasonable if they are connected with the electron-transfer chain.
Acta Crystallogr D Biol Crystallogr 1994 Sep 01
PMID:Location of active sites of NiFe hydrogenase determined by the combination of multiple isomorphous replacement and multiwavelength anomalous-diffraction methods. 1529 78

Complex [[(mu-SCH2)2N(4-NO2C6H4)]Fe2(CO)6] (4) was prepared by the reaction of the dianionic intermediate [(mu-S)2Fe2(CO)6](2-) and N,N-bis(chloromethyl)-4-nitroaniline as a biomimetic model of the active site of Fe-only hydrogenase. The reduction of 4 by Pd-C/H2 under a neutral condition afforded complex [[(mu-SCH2)2N(4-NH2C6H4)]Fe2(CO)6] (5) in 67 % yield. Both complexes were characterized by IR, 1H and 13C NMR spectroscopy and MS spectrometry. The molecular structure of 4, as determined by X-ray analysis, has a butterfly 2Fe2S core and the aryl group on the bridged-N atom slants to the Fe(2) site. Cyclic voltammograms of 4 and 5 were studied to evaluate their redox properties. It was found that complex 4 catalyzed electrochemical proton reduction in the presence of acetic acid. A plausible mechanism of the electrocatalytic proton reduction is discussed.
Chemistry 2004 Sep 20
PMID:Synthesis, structures and electrochemical properties of nitro- and amino-functionalized diiron azadithiolates as active site models of Fe-only hydrogenases. 1537 25

We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addition in the electrochemical cell of H(2), CO, or O(2) allow simple and virtually instantaneous determinations of the Michaelis constant, inhibition constant, or rate of inactivation, respectively, thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we determine and discuss how its affinity for the active site changes as the driving force for the H(+)/H(2) reaction is continuously varied. Inactivation by O(2) is a slow, bimolecular process (with pH-independent rate constant approximately 3 x 10(4) s(-1) M(-1) at 40 degrees C, under one atm of H(2)) that leads to a mixture of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H(2). This experimental approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.
J Am Chem Soc 2004 Sep 29
PMID:Inhibition and aerobic inactivation kinetics of Desulfovibrio fructosovorans NiFe hydrogenase studied by protein film voltammetry. 1538 53

Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.
Biophys J 2005 Sep
PMID:Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake. 1595 84

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.
Biophys J 2005 Sep
PMID:An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction. 1595 85

In nitrogen-limiting conditions, approximately 10% of the vegetative cells in filaments of the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 differentiate into nitrogen-fixing heterocysts. During the late stages of heterocyst differentiation, three DNA elements, each embedded within an open reading frame, are programmed to excise from the chromosome by site-specific recombination. The DNA elements are named after the genes that they interrupt: nifD, fdxN, and hupL. The nifD and fdxN elements each contain a gene, xisA or xisF, respectively, that encodes the site-specific recombinase required for programmed excision of the element. Here, we show that the xisC gene (alr0677), which is present at one end of the 9,435-bp hupL element, is required for excision of the hupL element. A strain in which the xisC gene was inactivated showed no detectable excision of the hupL element. hupL encodes the large subunit of uptake hydrogenase. The xisC mutant forms heterocysts and grows diazotrophically, but unlike the wild type, it evolved hydrogen gas under nitrogen-fixing conditions. Overexpression of xisC from a plasmid in a wild-type background caused a low level of hupL rearrangement even in nitrogen-replete conditions. Expression of xisC in Escherichia coli was sufficient to produce rearrangement of an artificial substrate plasmid bearing the hupL element recombination sites. Sequence analysis indicated that XisC is a divergent member of the phage integrase family of recombinases. Site-directed mutagenesis of xisC showed that the XisC recombinase has functional similarity to the phage integrase family.
J Bacteriol 2005 Sep
PMID:Heterocyst-specific excision of the Anabaena sp. strain PCC 7120 hupL element requires xisC. 1610 44

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.
Infect Immun 2005 Sep
PMID:Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice. 1611 46

The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.
Biochemistry 2005 Sep 13
PMID:Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB. 1614 21


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