A purification procedure for the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough, Na- MATERIALS AND METHODSGrowth of Organisms and Preparation of Crude Extract. 57Fe-labeled D. vulgaris (Hildenborough, NCIB 8303) was grown for 38 hr in lactate/sulfate medium containing the following components per liter: sodium lactate (60%), 12.5 ml; NH4Cl, 2 g; MgSO4-7H2O, 2 g; K2HPO4, 0.5 g; Na2SO4, 4 g; CaCl2 2H2O, 0.035 g; Na2S-9H20, 0.25 g; 57Fe (enrichment, 95%), 1 mg; EDTA, 2 mg; cysteine hydrochloride, 0.125 g; mineral solution (9), 1 ml. The pH of the medium was 7.2 after autoclaving. Cells (407 g) from 200 liters were harvested and then suspended in 400 ml of 10 mM Tris-HCl (pH 7.6) and frozen at -80°C for 2 days. The cells were then slowly defrosted for about 20 hr and were centrifuged at 19,000 x g for 45 min. The supernatant was called crude extract.Purification of Hydrogenase. All purification procedures were carried out in air, at 40C, and the pH of the buffers was 7.6 (measured at 50C). A summary of the results of a typical purification is presented in Table 1.First DEAE-Bio-Gel column. The crude extract was placed on a DEAE Bio-Gel column (6 x 32.5 cm) and washed with 500 ml of 0.01 M Tris-HCl buffer, and the proteins were eluted with two Tris-HCl linear gradients ( Hydroxylapatite column. The collected hydrogenase-containing fractions were loaded onto a hydroxylapatite (BioRad) column (4.5 x 24.5 cm) and the column was washed with 100 ml of 1 mM potassium phosphate (KP) buffer at a flow rate of 40 ml per hour. The protein was then eluted by two phosphate linear gradients (1.25 liters of 1 mM KP buffer and 1.25 liters of 0.2 M KP buffer, 625 ml of 0.2 M KP buffer and 625 ml of 0.3 M KP buffer). The hydrogenase activity was detected between 1650 ml and 2390 ml and the volume concentrated to 15 ml by using a diaflow apparatus with a YM 30 membrane.Second DEAE-Bio-Gel column. The concentrated protein solution was diluted 1:10 with 10 mM Tris-HCl and then applied to the second DEAE-Bio-Gel column (4.5 x 22.5 cm). The column was washed with 200 ml of 0.01 M Tris-HCl, and then a linear gradient was constructed (750 ml of 0.01 M Tris-HCl and 750 ml of 0.2 M Tris-HCI). The hydrogenase was collected between 1200 and 1330 ml and the volume was concentrated to 14 ml.Sephacryl S-200 column. The protein was loaded on a Sephacryl S-200 column (5.4 x 85 cm) and eluted with 0.05 M KP buffer at a flow rate of 20 ml/hr. The protein was collected in 5 ml-fractions between 650 and 700 ml. Purity of the hydrogenase was established by polyacrylamide disc electrophoresis (10) as well as NaDodSO4/acrylamide electrophoresis (11). The purified hydrogenase had a specific activity of 4800 ,umol of H2/min per mg of protein and an A400 nm/A280 nm ratio of 0.36.Assays and Metal Determination. Hydrogenase activity was determined by the H2 evolution assay (12). Hydrogen was determined by means of a Varian 4600 gas chromatograph (4) and protein, by the Bradford method (13) using bovine serum albumin as a reference st...

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We sequenced the Nil2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides.A significant fraction of the hydrogenase activity of sulfate-reducing bacteria belonging to the genus Desulfovibrio is localized in the periplasmic space (2,18,19). Two types of periplasmic hydrogenase have been isolated and characterized: a nickel-and nonheme iron-containing hydrogenase (nickel-iron hydrogenase) composed of two subunits (8,13) and an exclusively nonheme iron (three Fe4S4 clusters)-containing hydrogenase (iron hydrogenase) of higher specific activity (11,19). Voordouw et al. (22) were the first to provide evidence that the iron hydrogenase is also composed of two subunits (45.8 and 13.5 kilodaltons [kDa]). They cloned and sequenced a DNA fragment encoding a protein reactive with polyclonal antibody to the periplasmic iron hydrogenase of D. vulgaris (Hildenborough). The following conclusions were drawn based on the nucleotide sequence (23). The hydrogenase operon is composed of two genes, separated by 14 base pairs and coding for 46-and 13.5-kDa polypeptides, i.e., the large and small subunits. The NH2-terminal amino acid sequence of the large subunit is colinear with the AUG translational start of the gene, suggesting the absence of a signal peptide and leading to speculation that the gene might code for a cytoplasmic hydrogenase. A high degree of homology between the NH2-terminal region of the large subunit and the amino acid sequences of the eight iron ferredoxins substantiates the biochemical information, indicating that two of the three iron clusters are of the ferredoxin type (7, 11). As 10 of the remaining 11 cysteine residues were located on the large subunit, it was suggested that the third Fe4S4 cluster is also located on the large subunit; however, the possible involvement of the single cysteinyl residue on the small subunit as a ligand for an iron-sulfur cluster could not be eliminated.Because of the existence of multiple molecular forms of hydrogenase (1, 23), we were interested in establishing whether the periplasmic hydrogenase of D. vulgaris isolated by our purification procedure was identical to that encoded by the hydrogenase gene cloned by Voordouw and Brenner (21) and, if so, whether the small subunit of the iron hydrogenase is encoded by the gene immediately downstream from the gene for the 46-kDa protein. In this note we report that the NH2-terminal amino acid sequence of the large subunit of the iron hydrogenase isolated by our purification procedure corresponds to the nucleotide sequence of the gene (21) coding for the large subunit but that the small * Corresponding author.subunit lacks a hydrophobic NH2-te...

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