Localization of hydrogenase in Desulfovibrio gigas cells

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.     

Highly active immobilized hydrogenase from Desulfovibrio gigas

The hydrogenase from the sulfate reducer $\text{Desulfovibrio gigas}$ has been immobilized by covalent coupling onto a porous silica support. Two methods have been used: glutaraldehyde activation of aliphatic amino Spherosil and diazotation of aromatic amino Spherosil. The effect of cytochrome C₃ and CC₃ addition during coupling has been investigated. The highest enzymatic activity (4440 U/g support) and immobilization yield (29 %) was obtained when coupling hydrogenase in the presence of cytochrome C₃ or CC₃ with diazotized aromatic amino silica. This immobilized hydrogenase preparation which shows a very good resistance to oxygen inactivation seems suitable for hydrogen photoproduction by coupling with illuminated chloroplasts.     

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas.

The hydrogenase of the sulfate-reducer $\text{Desulfovibrio gigas}$ 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.     

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas.

We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).     

A novel membrane-bound Ech [NiFe] hydrogenase in Desulfovibrio gigas

In the present study, we report the identification of an operon with six coding regions for a multisubunit membrane-bound [NiFe] hydrogenase in the genome of Desulfovibrio gigas. Sequence analysis of the deduced polypeptides reveals a high similarity to subunits of proteins belonging to the family of Ech hydrogenases. The operon is organised similarly to the operon coding for the Ech hydrogenase from Methanosarcina barkeri, suggesting that both encode very similar hydrogenases. Expression of the operon was detected by Northern blot and RT-PCR analyses, and the presence of the encoded proteins was examined by Western blotting. The possible role of this hydrogenase is discussed, relating it with a potential function in the H(2) cycling as a mechanism for energy conservation in D. gigas. The present study provides therefore valuable insights into the open question of the energy conserving mechanism in D. gigas.     

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.     

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from $\text{Desulfovibrio}\text{gigas}$is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ～2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.     

Reactivation of the hydrogenase from Desulfovibrio gigas by hydrogen. Influence of redox potential

The specific activity of the periplasmic hydrogenase from Desulfovibrio gigas is increased approximately 10-fold in the H2 utilization assay with benzyl viologen by several hours of incubation under an atmosphere of H2. After a variable lag phase during which residual traces of O2 are removed, the reversible activation is exponential. The extent of activation is dependent on pH and the redox potential of the incubation medium. A tentative model based on the existence of a monoelectronic redox center is proposed as shown in the following equation: (formula; see text) The potential of this redox couple was determined to be -310 mV (pH = 7; T = 298 K) versus the normal hydrogen electrode.     

Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy

We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed.     

ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase

The electron spin resonance (ESR) spectra of hydrogenase from Desulfovibrio gigas were observed during the activation of the enzyme in the oxidized, ‘unready’, state by hydrogen. Signals from nickel(III) (Ni-A), and the [3Fe-xS] cluster were reduced within less than 5 min, and a broad ESR signal appeared at the same time. On the basis of simultaneous changes in optical absorption spectrum, it is proposed that the broad ESR signal represents one or possibly both [4Fe-4S] clusters in the reduced state. The increase of enzyme activity was much slower (at 20°C), and was accompanied by the appearance of another type of nickel signal (Ni-C), and a further small decrease and the Ni-C signal became more intense. On further reoxidation by the dye dichlorophenolindophenol at pH above 7.0 the enzyme was converted to the ‘ready’ state, which could now be reactivated much more rapidly by strong reductants. The proportion of the ready state correlated with a third type of nickel signal, Ni-B. The unready enzyme could also be slowly activated by milder reducing conditions which reduced Ni-A and the [3Fe-xS] cluster but did not induce significant amounts of the Ni-C and [4Fe-4S]¹⁺ signals. The optical absorption changes indicate that the Ni-A is not coupled to an iron-sulphur cluster. It is proposed that the activation of the enzyme involves reduction of the nickel and possibly iron-sulphur centres, followed by a conformational change which alters the coordination state of nickel, and that the unready state contains Ni(III) in the inactive conformation, the ready state Ni(III) in the active conformation, and the active state Ni(I).     

Nickel controls the reversible anaerobic activation/inactivation of the Desulfovibrio gigas hydrogenase by the redox potential

An electrochemical method of hydrogenase activity measurement is developed. It permits a new approach to the activation/inactivation process of the Desulfovibrio gigas hydrogenase. A monolayer of hydrogenase is grafted onto a glassy carbon electrode which is both the support of the enzyme and the detector of the activity. The physicochemical composition of the enzyme microenvironment is thus well defined and easily controlled by the electrode potential. Successive periods of inactivation and activation are applied to the same hydrogenase molecules, thus the activity can be correlated to the chronology of the experiments. We distinguish two kinds of activation/inactivation processes. The first one, already described for the enzyme stored for some months in aerobic conditions, is a slow activation by molecular hydrogen or a reducing medium (half-reaction time = 2 h). The second one is an anaerobic inactivation by an oxidizing potential. This first order inactivation (half-reaction time = 10 min) is fully reversible. This modulation of the activity level is controlled by an Ni(III)/Ni(II) redox couple (Eh = -455 mV/calomel-saturated KCl electrode at pH 8.3) involving one electron and one proton. This work proposes an explanation for the activation of the hydrogenase taking into account the participation of an [Fe-S] cluster and of the nickel atom.     

Isolation, amino acid analysis and N-terminal sequence determination of the two subunits of the nickel-containing hydrogenase of Desulfovibrio gigas

The two subunits of the nickel-iron hydrogenase from Desulfovibrio gigas have been purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and their amino acid compositions have been determined. The N-terminal sequences for 15 residues of the large subunit (Mr 62,000) and 25 residues of the small subunit (Mr 26,000), respectively, were established. The occurrence of several cysteine residues in the small subunit is discussed in relation with their possible role in the binding of the redox centers of the enzyme.     

Magnetic susceptibility of hydrogenase from Desulfovibrio vulgaris

Magnetization and magnetic susceptibility measurements revealed that the hydrogenase [EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki F has an independent unpaired electron in its iron-sulfur cluster. The paramagnetic center of the Desulfovibrio hydrogenase is, therefore, different from that in the Chromatium hydrogenase which interacts with another paramagnetic center, probably nickel.     

Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.