Yves Berlier

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.

Inhibition studies of three classes of Desulfovibrio hydrogenase: application to the further characterization of the multiple hydrogenases found in Desulfovibrio vulgaris Hildenborough

The three types of hydrogenase hitherto characterized in genus Desulfovibrio exhibit distinctive inhibition patterns of their proton-deuterium exchange activity by CO, NO and NO2-. The (Fe) and (NiFeSe) hydrogenases are the most sensitive to all three inhibitors while the (NiFe) enzymes, relatively little inhibited by CO, are still very sensitive to NO but unaffected by NO2-. These differences together with some specific catalytic properties, in particular the pH profile and the H2 to HD ratio in the exchange reaction, constitute a simple means of characterizing multiple hydrogenases present in one or different species.

The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria

Different patterns have been found in the pH dependence of hydrogenase activity with enzymes purified from different species of Desulfovibrio. With the cytoplasmic hydrogenase from Desulfovibrio baculatus strain 9974, the pH optima in H2 production and uptake were respectively 4.0 and 7.5 with a higher activity in production than in uptake. The highest D2-H+ exchange activity was found also at pH 4.0 but the optima differed for the HD and the H2 components. Both similarly rose when the pH decreased from 9.0 to 4.5, but the rate of H2 evolution slowed whereas the HD evolution continued rising till pH values around 3.0 were reached. The H2 to HD ratio at pH above 4.5 was higher than one. With the periplasmic hydrogenase from Desulfovibrio vulgaris Hildenborough, the highest exchange activity was near pH 5.5, the same value as in hydrogen production. The periplasmic hydrogenase from Desulfovibrio gigas had in contrast the same pH optimum in the exchange (7.5-8.0) as in the H2 uptake. The ratio of H2 to HD was below one for both enzymes. These different patterns may be related to functional and structural differences in the three hydrogenases so far studied, particularly in the composition of their catalytic centers.

A proton-deuterium exchange study of three types ofDesulfovibrio hydrogenases

SummaryHydrogenases are among the main enzymes involved in bacterial anaerobic corrosion of metals. The study of their mode of action is important for a full comprehension of this phenomenon. The three types ofDesulfovibrio hydrogenases [(Fe), (NiFe), (NiFeSe)] present different patterns in the pH dependence of their activity. The periplasmic enzyme fromDesulfovibrio salexigens and the cytoplasmic enzyme fromDesulfovibrio baculatus both have pH optima at 7.5 for H2 uptake and 4.0 for H2 evolution and H+−D2 exchange reaction (measured by membrane-inlet mass-spectrometry). The H2 to HD ratio at pH above 5.0 is higher than 1.0. The periplasmic hydrogenase fromD. gigas presents the same pH optimum (8.0) for the H+−D2 exchange as for H2 consumption. In contrast, the enzyme fromD. vulgaris has the highest activity in H2 production and in the exchange at pH 5.0. Both hydrogenases have a H2-to-HD ratio below 1.0.

Effect of pH on H-2H exchange, H2 production and H2 uptake, catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

Abstract (1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H2H, H2 or 2H2 produced when the enzyme catalyses the exchange between 2H2 and H2O or H2 and 2H2O. (2) In the 2H2-H2O system the measured rate of H2 production was always higher than that of H2H. The H 2 H 2 H ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H2 with respect to H2H is consistent with the hypothesis of a heterolytic cleavage of 2H2 into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H2-2H2O system, the exchange rate was much lower than in the 2H2-H2O system, indicating a marked isotopic effect of 2H2O. (4) The H-2H exchange activity, determined from the initial velocity of H2H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H2 production while at pH 8.3–8.5 there is a maximum of H2 oxidation activity. (5) In ordinary H2O the Km for hydrogen uptake estimated either from H2 consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H2 and 2H2 indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H2O to 2H2O does not affect the Km of the enzyme for H2 but lowers the Vmax value about 10-fold. The Km for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D2 uptake and in H2 and HD evolution. In contrast to the nitrogenase-mediated H2 production, this anaerobic H2 and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H2 and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D2 is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.

A gas chromatographic-mass spectrometric technique for studying simultaneous hydrogen-deuteron exchange and para-orthohydrogen conversion in hydrogenases of Desulfovibrio vulgaris Hildenborough

An original gas chromatographic-mass spectrometric technique is described for studying simultaneous dihydrogen-deuteron exchange and para-ortho H2 conversion catalyzed by different Desulfovibrio hydrogenases. Para and orthohydrogens are separated on an alumina column at the temperature of liquid nitrogen, but if both HD and ortho H2 are present, their retention times are too close to each other for total separation and only one peak is observed with a thermal conductivity detector. In order to resolve the peaks from one another, a fraction of the gas released from the gas chromatograph column is admitted to the ion source of a mass spectrometer, where the gases are separated according to their respective masses. Because of a peak-jumping system, the different components involved in the exchange and in the conversion reactions can be scanned so that the spectra corresponding to mass m/e 2 (para and ortho H2), m/e 3 (HD), and m/e 4 (D2) can be obtained simultaneously. This technique has been employed to resolve a controversial problem concerning the occurrence or lack of any para-orthohydrogen conversion in heavy water. Actually both exchange and conversion were demonstrated to occur with a (NiFe) hydrogenase, whereas with a (NiFeSe) hydrogenase, which had an exchange activity equivalent to that of the former, practically no para-ortho conversion could be observed in D2O. These findings are related to the constitutional and catalytic properties of the hydrogenases belonging to the different classes.

The activation of the periplasmic (NiFe) hydrogenase of Desulfovibrio gigas by carbon monoxide

The activation of the periplasmic (NiFe) hydrogenase from Desulfovibrio gigas by dihydrogen is a complex phenomenon involving both ‘slow’ and ‘fast’ reactions. Carbon monoxide, a competitive inhibitor of hydrogenase activity, is demonstrated to cause the slow activation nearly as well as dihydrogen. Carbon monoxide does not reduce the (NiFe) hydrogenase and the fast reductive activation is effected by deuterium in the exchange assay. In the presence of dithionite, which immediately reduces the redox centers of the (NiFe) hydrogenase, the slow activation is still essential to attain full activity. Thus, the slow non‐reductive and fast reductive steps of the activation can occur in any sequence.

The relationship between hydrogen metabolism, sulfate reduction and nitrogen fixation in sulfate reducers

SummaryHydrogenase and nitrogenase activities of sulfate-reducing bacteria allow their adaptation to different nutritional habits even under adverse conditions. These exceptional capabilities of adaptation are important factors in the understanding of their predominant role in problems related to anaerobic metal corrosion. Although the D2−H+ exchange reaction indicated thatDesulfovibrio desulfuricans strain Berre-Sol andDesulfovibrio gigas hydrogenases were reversible, the predominant activity in vivo was hydrogen uptake. Hydrogen production was restricted to some particular conditions such as sulfate or nitrogen starvation. Under diazotrophic conditions, a transient hydrogen evolution was followed by uptake when dinitrogen was effectively fixed. In contrast, hydrogen evolution proceeded when acetylene was substituted as the nitrogenase substrate. Hydrogen can thus serve as an electron donor in sulfate reduction and nitrogen metabolism.

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.