Further characterization of the [Fe]‐hydrogenase from Desulfovibrio desulfuricans ATCC 7757

Abstract

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G. and Martin, S. M. (1980) Can. J. Microbiol. 26,1214-12231 were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfnvibrio vulgaris Hildenborough [Fel-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acidlabile sulfur/molecule and had ~4 0 0 , 52.5 mM-l . cm-l. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H,-uptake and 2H2/'H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4SI1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H,-activating site in other [Fel-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fel-hydrogenase, similar in most respects to the well characterized [Fel-hydrogenase from D. vulgaris Hildenborough.Hydrogenase, an enzyme widely distributed among microorganisms, catalyzes the reversible oxidation of molecular hydrogen. Biochemical studies have clearly established the existence of two main groups of enzymes in sulfate-reducing bacteria of the genus Desulfovibrio, the one containing either nickel or nickel and selenium atoms in addition to iron/sulfur clusters ([NiFeI-hydrogenases and [NiFeSeI-hydrogenases) and the other containing exclusively iron-sulfur clusters ([Felhydrogenases). These hydrogenases differ in their metal-centre composition, mechanistic properties, sensitivity to inhibitors, amino acid sequences and immunological properties [ 11.In contrast to the widespread nickel-containing hydrogenases, the known [Fel-hydrogenases, which exhibit high catalytic activity, form a small family of proteins [2]. Among these enzymes, the periplasmic [Fel-hydrogenase from Desulfovibrio vulgaris Hildenborough has been thoroughly investigated by a variety of biochemical and spectroscopic techniques [3 -111. The protein exhibits a molecular mass of 56 kDa and is composed of two different subunits (46 kDa and 10 kDa).Correspondence to E. C . ...

A positive role for hydrogen gas in adventitious root development

“…[4][5][6] Some studies found that H 2 and hydrogenase activity could be detected in higher plants. 7,8 The H 2 production was also reported in Medicago sativa and Arabidopsis.…”

Spin distribution of the H-cluster in the Hox–CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions

“…The isolation followed the purification protocol of Hatchikian [53], originally established by van der Westen [54]. The wet cell pellet (46 g) of a harvested D. desulfuricans was incubated for 30 min at room temperature in a buffer with 50 mM Na 2 -EDTA and 50 mM tris (hydroxymethyl)aminomethane (Tris)/HCl (adjusted to pH 9 with HCl) under gentle stirring to obtain the periplasmic fraction, which contains the [FeFe] hydrogenase.…”

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.…”