EPR of a novel high-spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728-3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 spin/mol. In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 spin/mol) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with geff = 5.0 which, under the assumptions that S = 2 and [delta ms] = 2, quantitates to roughly one spin/mol. Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin-state transition of an iron-sulfur cluster.

Application of hydrogenase in biotechnological conversions.

Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented.

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough).

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.

Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli.

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.