Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.     

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).     

The effect of histidine residue modification on tyrosinase activity and conformation: inhibition kinetics and computational prediction

We found that the histidine chemical modification of tyrosinase conspicuously inactivated enzyme activity. The substrate reactions with diethylpyridinecarbamate showed slow-binding inhibition kinetics (K(I) = 0.24 +/- 0.03 mM). Bromoacetate, as another histidine modifier, was also applied in order to study inhibition kinetics. The bromoacetate directly induced the exposures of hydrophobic surfaces following by complete inactivation via ligand binding. For further insights, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and diethylpyridinecarbamate. The docking simulation was shown to the significant binding energy scores (-3.77 kcal/mol by AutoDock4 and -25.26 kcal/mol by Dock6). The computational prediction was informative to elucidate the role of free histidine residues at the active site, which are related to substrate accessibility during tyrosinase catalysis.     

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.     

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.     

Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The nucleotide sequence of the 4.7-kb SalI/EcoRI insert of plasmid pHV 15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain-termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH2-terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N-formylmethionine residue precedes the NH2-terminal amino acid Ser-1. There is no evidence for a leader sequence. The NH2-terminal part of the hydrogenase shows homology to the bacterial [8Fe-8S] ferredoxins. The sequence Cys-Ile-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro-Xaa-Xaa-Ala-(Ile) occurs twice both in the hydrogenase and in [8Fe-8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe-4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996]. These results, therefore, suggest that two electron-transferring ferredoxin-like [4Fe-4S] clusters are located in the NH2-terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46-kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5-kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5-kDa polypeptide in addition to the 46-kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two-subunit enzyme.     

On the reaction of papain and succinylpapin with diazo-1-H-tetrazole.

1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.     

The role of histidine residues of formate dehydrogenase from Bacterium sp. 1]

The reaction between formate dehydrogenase from Bacterium sp. 1 and diethylpyrocarbonate results in the enzyme inactivation. 4 histidine residues can be blocked per subunit by this reagent. The enzyme activity correlates with the disappearance of free histidines. The process of enzyme inactivation is biphasic and obeys pseudo-first-order kinetics. NAD and NADH slow down the rate of inactivation, but do not protect histidine residues against modification. Formate does not protect the enzyme. The modification of 80% of histidines increases the Km value for both substrates 3-fold. The general conformation of enzyme in the course of modification is preserved. The modification of histidines markedly decreases the reactivity of an essential SH-group of formate dehydrogenase against the Ellman reagent.     

Conformational changes in the membrane-bound hydrogenase of Bradyrhizobium japonicum. Evidence that the redox state of the enzyme affects its accessibility to protease and membrane-impermeant reagents

The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

Redox interaction of cytochrome c3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric cytochrome c3 except N-terminus, Pro, and Gly73 were assigned. 1H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome c3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c3. To investigate the transient complex of cytochrome c3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the Km of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.