Desulfovibrio vulgaris hydrogenase: a nonheme iron enzyme lacking nickel that exhibits anomalous EPR and Mössbauer spectra.

Abstract

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...

The relationship between activity and the axial g=2.06 EPR signal induced by CO in the periplasmic (Fe) hydrogenase from Desulfovibrio vulgaris

“…This signal is characteristic of a three-iron center but, because of its low spin quantitation, does not appear to be of catalytic signifisignificance. There is no effect of CO on this EPR signal (g= 1.94) which is characteristic of two interacting (Fe&,) centers and Mossbauer data support this conclusion [3,14]. Variable amounts (0.03 spin/molecule) of an axial g= 2.06, 2.01 EPR signal may be distinguished in the 6.5 K spectrum of the hydrogen-reduced hydrogenase [3,5] and, above 20 K, the g= 2.06 signal becomes the only EPR feature observed [3].…”

“…The native (Fe) hydrogenase shows a weak g= 2.02 isotropic EPR signal which integrates to approx. 0.02 spin/molecule and has been reported previously [3,4]. This signal is characteristic of a three-iron center but, because of its low spin quantitation, does not appear to be of catalytic signifisignificance.…”

“…56 kDa and is composed of two subunits (46 kDa, 10.5 kDa) [1,2]. Spectroscopic and cluster-extrusion data indicate the prosthetic groups to be two ferredoxin-type [4Fe-4S] clusters and an iron cluster with abnormal spectroscopic properties [3,4]; however, the total iron content has been reported to vary between 11 and 16 atoms per molecule [3,5]. The unique sensitivity of the hydrogenase activity found in D. vulgaris to CO was first recognized by Krasna and Rittenberg [6] but its significance was not appreciated until the presence of multiple hydrogenases with different biochemical properties in this bacterium was reported [7].…”

Nickel hydrogenases: in search of the active site

“…From several bacteria hydrogenases have been purified in which no other metal than Fe could be detected (Acetobacter woodii [156], Acetomicrobium flavidum [169], Clostridium pasteurianum [3,41], Desulfovibrio desulfuricans ATCC 7757 [93], possibly Desulfovibrio salexigens [64], D. vulgaris [103,205], Megasphaera elsdenii [74], Thermatoga maritima [108] and Trichomonas vaginalis [152]). Hence these enzymes clearly belong to a separate class.…”

Monovalent nickel in hydrogenase from Chromatium vinosum

“…In many of these hydrogenases the presence of nickel has been established [4-151. In some cases, however, Ni has proved to be absent [16,17]. In the nickel enzymes both nickel and at least one Fe-S cluster seem to be required for activity [8-141, except possibly in the enzyme from Methanobacterium thermoautotrophicum, strain Marburg [4,5].…”