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 (z 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 c$ 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.The cloning and nucleotide sequence of the genes encoding the hydrogenase from Desulfovibrio vulgaris H indicated this protein to be an c$ dimer [l, 21 and this has been confirmed by biochemical studies [3]. A prerequisite for further advances in the study of this enzyme is that it can be expressed at high levels in active form in a suitable host, preferably Escherichia coli. In this article the modification of the parent plasmid pHV1.5 to plasmids that direct a high level of expression of hydrogenase polypeptides in E. coli is described and the purification of an inactive derived hydrogenase is reported. The characterization of the purified protein clearly indicates the nature of the defect that prevents the recombinant enzyme to be enzymatically active.We consider the present work to be a first step towards the active expression of D. vulguris H hydrogenase in E. coli. MATERIALS AND METHODS MaterialsRestriction endonucleases, T4 DNA ligase, T4 DNA polymerase and DNA polymerase (Klenow fragment) were obtained from the same suppliers as described previously [l, 21. 12sI-labelled protein A (30 mCi/mg), [ u -~~P I~A T P ,

The subunit composition and Fe/S content of nine different batches of D. vulgaris (H) hydrogenase have been determined. SDS-PAGE and FPLC show the enzyme to be an u/3 dimer consistent with the molecular mass of 46 + 13.5 kDa recently inferred from the nucleotide sequence. Based on this molecular mass and a protein determination calibrated on tryptophan + tyrosine content, the enzyme is found to contain 14-16 Fe and 12-14 Sz-, and Ed,, = 45 mM-lcm-r.It is suggested that the active site of this bidirectional hydrogenase is not a [4Fe-4S] cluster, but rather is a novel cluster comprised of approx. 6 Fe and 6 Sz-. HvdrogenaseIron-sulfur cluster (Desulfovibrio vulgaris)

The EPR of reoxidized hydrogenase from Des~ifuvi~rio ~lgar~ (H.) has been reinvestigate.In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728-37321 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 Idm,l = 2, quantitates to roughly one spin/mol. Ethylene glycoi affects the relative intensity of the different signals. It is suggested that 0, sensitization parallels a spin-state transition of an iron-sulfur cluster. Hydrogenase (Desulfovibrio vulgaris)rron-su~ur cluster

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 (MeZSOj. Hydrogen consumption activity does not change up to 30 % (v/v) of MezSO. 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.Studies in this laboratory on the kinetic properties of the hydrogenase isolated from Megasphaera elsdenii have been reported in a recent series of articles [I -41. It has been found [l] that H2 production activity of M . elsdenii hydrogenase does not follow Michaelis-Menten kinetics with respect to its donors. The kinetics for M . elsdenii enzyme have been explained as being the result of a random mechanism, applicable to both the natural donors, flavodoxin and ferredoxin, as well as to the artificial donor, methyl viologen semiquinone. This deviation from Michaelis-Menten kinetics was first observed for the natural donors. The mathematical model proposed was then tested on the artificial donor methyl viologen. Since the deviation becomes more pronounced at higher methyl viologen semiquinone concentrations, the dimer of the semiquinone had to be taken into account. Therefore the methyl viologen experiments were performed at different salt concentrations since the association constant of the monomer-dimer equilibrium is dependent on salt. It was shown that the enzyme shows different kinetics with the monomer compared to the dimer. Also, salt influences the kinetics, not only by shifting the dimer equilibrium, but also through its effect on the protein itself [2]. A similar salt stimulation has been found by Roessler and Lien [5] for another hydrogenase, isolated from Chlamydomonas reinhardtii. The activity of the M . elsdenii enzyme is furthermore stimulated at low pH, which is a resultant of the effect of pH on the enzyme itself and the higher H + (substrate) concentration [4]. The midpoint potential of the enzyme as derived from initial activity measurement shows the same potential as the hydrogen electrode, at the pH tested, which allows the enzyme to be reduced at relatively low pH. The influence of dimethylsulfoxide (MezSOj and ethylene glycol were also measured to determine whether or not organic solvents influence the enzyme in a way different from salt.Since the hydrogenase isolated from Desulfovihrio vulgaris is a highly active enzyme and also contains 12 iron and Abbreviations. Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; cy...

Upon reduction of the oxygen‐stable hydrogenase of Desulfovibrio vulgaris strain Hildenborough this enzyme becomes oxygen sensitive. Only anaerobic oxidation of the reduced enzyme with 2,6‐dichlorophenol‐indophenol, in the presence of iron and EDTA, followed by addition of Tris‐EDTA (pH 9.0) converts the enzyme back into its oxygen‐stable state, without loss of activity.