J. Le Gall

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas

Abstract The hydrogenase of the sulfate-reducer Desulfovibrio gigas has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H2 evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe4S4 clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.

Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors

A purified preparation of hydrogenase from D. gigas was inactive toward ferredoxin, flavodoxin or rubredoxin in the absence of cytochrome c3 (M.W. 13,000), in an atmosphere of hydrogen, although direct reduction of benzyl viologen or FMN was possible. The hydrogen evolution reaction from dithionite was possible with methyl viologen. The same reaction also occured with cytochrome c3 (M.W. 13,000) or cytochrome c3 (M.W. 26,000). Addition of either ferredoxin or flavodoxin did not accelerate the reaction.

Chemical study of two flavodoxins extracted from sulfate reducing bacteria

In this paper is presented a preliminary study of the structure of two sulfate reducing bacteria flavodoxins: Desulfovibrio gigas and Desulfovibrio vulgaris. Their respective spectroscopic characteristics have been investigated together with their amino acid composition and the nature of the flavin linked to the protein. This co-factor, that has been shown to be FMN, can be splitted off easely. In the case of the flavodoxin from D.gigas, reassociation between apoprotein and FMN is described. The two flavodoxins are very similar to the flavodoxins described from other organisms.

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli: A mass spectrometric study

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Isolation and characterization of desulforedoxin, a new type of non-heme iron protein from Desulfovibrio gigas

Abstract Desulfovibrio gigas desulforedoxin is a new type of non-heme iron protein of molecular weight 7,900. It contains two iron atoms, no labile sulfide and eight cysteine residues per molecule. The optical spectrum of the oxidized form presents important differences from that of the rubredoxin type proteins. Upon reduction with dithionite there is no contribution to the visible region. The oxidation-reduction process is reversible but the protein is sensitive to repeated redox cycles. The protein contains 73 amino-acid residues. Like rubredoxin it does not contain histidine and arginine but it also lacks proline, isoleucine, phenylalanine, and tryptophan. The N-terminal sequence has been determined up to 35 residues and no evident homology was found with other non-heme iron proteins.

Electron paramagnetic resonance and light absorption studies on c-type cytochromes of the anaerobic sulfate reducer desulfovibrio

Abstract 1. 1. EPR and optical studies provide for differentiation of the c-type cytochromes examined into three groups with different heme contents. The three groups consist of: the mono-heme type, cytochrome c-553; cytochromes c3 and c′3 containing at least two hemes per molecule; and cytochromes cc3 and cc′3 which contain at least four hemes per molecule. This division into three groups agrees with a previous similar grouping based on amino acid composition and/or sequence. 2. 2. EPR studies suggest that heme-heme interaction is manifest in the ferric state of cytochrome c3. At about a half-reduced state, an EPR-detectable intermediate with a decreased degree of interaction between hemes is observed. 3. 3. Based on EPR and light absorption changes, cytochrome c3 is extremely stable in 8 M urea in the ferric state. However, repeated reduction and reoxidation in the presence of 8 M urea results in the apparent conversion of the multi-heme system to a mono-heme system. 4. 4. These and other observations suggest a conformation change involving a possible reorientation of the multi-heme moieties in the partially or fully-reduced tate. Depending on the reactant(s) present this process may be reversible.

A molybdenum-containing iron-sulphur protein from desulphovibrio gigas

Summary A protein that contains molybdenum, iron and labile sulphide has been isolated from Desulphovibrio gigas . The molecular weight was estimated to be approx. 120,000, with 20 atoms of iron, 20 of labile sulphide, and 1 of molybdenum per molecule. The protein is acidic, with isoelectric point 4.1 ± 0.1. The iron-sulphur chromophores are of the 2Fe-2S type, as indicated by optical absorption and circular dichroism spectra, and EPR spectra of the protein in 80% dimethylsulphoxide. EPR spectra show the presence of two types of iron-sulphur centre in the molecule.

EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris.

EPR spectroscopy in conjunction with oxidation-reduction potentiometry has been used to determine the half-reduction potentials of the four hemes of cytochrome c3. As predicted, the four hemes of cytochrome c3 have different mid-point potentials. The Em values are: Heme I,--284 mV; Heme II,--310 mV; Heme III,--324 mV and Heme IV,--319 mV. The n-values in each case was near one.

Evidence for the presence of a b-type cytochrome in the sulfate-reducing bacterium Desulfovibrio gigas, and its role in the reduction of fumarate by molecular hydrogen

Abstract 1. The H2:fumarate reductase activities of particulate fractions prepared from Desulfovibrio gigas grown on various media (lactate-sulfate, fumarate or fumarate-sulfate) were measured. Activity was higher in the particulate fraction prepared from cells grown on fumarate or fumarate-sulfate media than in those prepared from cells cultivated on lactate-sulfate. 2. Various inhibitors were tested for their effect on fumarate reduction by H2 in a particulate fraction of Desulfovibrio gigas. A strong inhibitory effect was observed with 2-hetyl-4-hydroxyquinoline-N-oxide and antimycin A. These compounds act as inhibitors of an intermediary electron carrier between hydrogenase and fumarate reductase. 3. The strict anaerobe Desulfovibrio gigas has been found to contain a b-type cytochrome linked to the particulate H2:fumarate reductase activity. A protoheme obtained by extraction from the particulate fraction was characterized by its reduced pyridine hemochromogen.

Activation, reduction and proton-deuterium exchange reaction of the periplasmic hydrogenase from Desulfovibrio gigas in relation with the role of cytochrome c3.

The enzyme hydrogenase catalyzes the reversible oxidoreduction of the dihydrogen molecule, according to the reaction: Ha * 2H’ t 2e[ 11. Depending on the microorganisms and on the environmental conditions [2] the overall balance of the reaction can be directed towards either the production or the consumption of hydrogen gas. With the purified enzyme it is always possible with an adequate electron donor or acceptor to let the reversible reaction go in one or the other direction but the activity exhibited strongly depends upon the nature of the redox agent, especially its potential, and of the medium conditions [3]. The H+-D2 (or D’-HZ) exchange reaction is part of the reversible activity of hydrogenase [4] and provides an intrinsic measure of this activity. Since the overall balance is nil the exchange should proceed in the absence of any electron donor or redox substance. Yet, whereas the reaction readily takes place with the living cells [S], crude extracts [6] or even partly purified hydrogenases [7], the isolated enzyme in contrast has to be first activated for the exchange to proceed. This is generally achieved by addition of dithionite [6] or, in the case of Desulfovibrio, of the specific cytochrome c3 [8]; however both agents may interfere in several ways with the exchange reaction. The problem arises from the involvement in the loss and recovery of hydrogenase activity of different processes such as oxidation and reduction, oxygenation and deoxygenation. Dithionite for instance scavenges oxygen from the medium and from the enzyme centers and at the same time reduces hydrogenase [6]. Moreover, its action is altered according to the pH conditions [9]. As for cytochrome c3 it can act once reduced as a simple oxygen scavenger or, as it had been reported for Desuffovibrio vulgaris Miyazaki [8,10], it can accelerate both Hz evolution and the exchange reaction. On the contrary such a stimulation of Hz evolution has not been observed with Desulfovibrio gigas hydrogenase after addition of cytochrome c3 from that same organism [ Ill. It was therefore of interest to check whether in the case of the latter species cytochrome c3 had an effect upon the exchange reaction. This work shows that with the purified periplasmic hydrogenase from D. gigas the addition of an electron carrier or of a reducing agent was unnecessary either for the exchange reaction or for the activation of the enzyme. In the latter process, two successive steps were observed: (1) Requiring that oxygen be excluded from the enzyme centers but also probably implying a modification in these centers; this step could be achieved by physical or chemical means and was only accelerated in the presence of dithionite or cytochrome c3. (2) Consisting in a spontaneous reduction of the enzyme under molecular hydrogen and following the same kinetics whether cytochrome c3 was present or not.