(ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H 2, most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H 2͞CO2, the reduction of CO2 to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H 2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U´mg protein 21 at pH 7.0 and exhibited an apparent K m for the ferredoxin of 1 mm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U´mg protein 21 at pH 7.0. The apparent K m for the reduced ferredoxin was 7.5 mm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent K m for H 2 was 5 mm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H 2 /CO 2 -grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H 2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO 2 during growth on acetate, and to ferredoxin-linked H 2 uptake coupled to the reduction of CO 2 to the redox state of CO during growth on H 2 /CO 2 or methanol.Keywords: Methanosarcina barkeri; hydrogenase; complex I; energy conservation; iron±sulfur protein; ferredoxin.Methanosarcina barkeri is a methanogenic archaeon that can utilize H 2 /CO 2 , methanol or methylamines as energy substrates (reviewed in [1±3]). Three different [NiFe] hydrogenases have been characterized from Methanosarcina species: F 420 -reducing hydrogenase, F 420 -nonreducing hydrogenase and Ech hydrogenase.F 420 -reducing hydrogenase has been purified and characterized from M. barkeri [4,5]. The enzyme catalyzes the reduction of coenzyme F 420 which plays an important role as redox carrier in methanogenic archaea. The genome of M. barkeri contains two gene clusters ( frh and fre) encoding two related F 420 -reducing hydrogenases indicating the presence of two isoenzymes. Both operons were transcribed during growth on H 2 /CO 2 , methanol or trimethylamine [6]. In acetategrown cells no transcripts of these operons were detectable although these cells contain small amounts of F 420 -reducing hydrogenase activity (about 5% of the activity detectable in methanol-grown cells; Meuer and Hedderich, unpublished results).F 420 -nonreducing hydrogenase has been purified from M. barkeri and from Methanosarcina mazei. The purified enzyme was fo...

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe)4S] clusters. Two of the clusters could be reduced under 101 kPa of H 2 , whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe)4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.Keywords: Ech; hydrogenase; iron-sulphur; pH dependence; redox properties.Hydrogenases catalyse the simplest chemical reaction in nature: H 2 « 2H + + 2e -. They are found in wide variety of microorganisms. Hydrogenases enable some organisms to use H 2 as a source of reducing equivalents under both aerobic and anaerobic conditions. In other organisms the enzyme is used to reduce protons to H 2 , thereby releasing the reducing equivalents obtained from the anaerobic degradation of organic substrates [1,2]. On basis of the transition-metal content, hydrogenases can be divided into two major classes The study of hydrogenases in methanogens led to the discovery of a third class of hydrogenases, not containing any metals [9]. This class of enzyme is active only in the presence of its second substrate, N 5 ,N 10 -methenyltetrahydromethanopterin. There is evidence for an unknown nonmetal prosthetic group in this enzyme [10,11]. Methanogens also contain [NiFe]-hydrogenases and the expression of the several enzymes depends on the available energy sources [12,13]. Some time ago a membrane-bound [NiFe]-hydrogenase was isolated from methanogenic archaea [14], which consists of six subunits much like hydrogenase-3 of Escherichia coli. Hydrogenase-3 in E. coli is part of the formate-hydrogen lyase complex and is composed of seven different subunits [15]. This hydrogenase shows surprisingly little sequence homology with other [NiFe]-hydrogenases, except for the conserved residues coordinating the active site and the proximal Fe-S cluster. The enzyme showed a high sequence similarity with the CO-induced hydrogenase of Rhodospirillum rubrum [16,17]. The latter bacterium can grow anaerobically on CO and its [NiFe]-hydrogenase is thus expected to be insensitive towards CO. The same is expected for the ÔE. coli-like hydrogenaseÕ (Ech) from Methanosarcina barkeri [14,18]. From growth characteristics of R. rubrum and from cell-suspension experiments with M. barkeri, it can be inferred that the ...