Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri

Abstract

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...

Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation

“…Biochemical studies indicate that a 2[4Fe-4S] ferredoxin is the physiological substrate for Ech, but the physiological function of Ech has yet to be established. RNA hybridization and immunoblotting experiments indicate that the ech operon is expressed at similar levels under all conditions examined, suggesting only that it is required under many growth conditions (7,8).…”

“…From a biochemical perspective, the most thoroughly studied member of the family is the recently identified Ech found in the methanogenic archaeon M. barkeri (8). This enzyme catalyzes the reversible reduction of ferredoxin by H 2 and is the only family member that has been purified to homogeneity.…”

“…First, because the enzyme is an integral membrane protein related to complex I, it has been suggested that Ech catalyzes an energyconserving reaction. The oxidation of the carbonyl group of acetylCoA to CO 2 and H 2 , a partial reaction in the production of methane from acetate (the acetoclastic pathway), has been proposed as the Ech-dependent coupling site (8). This reaction is coupled to the generation of a proton motive force in M. barkeri (9).…”

“…This reaction is coupled to the generation of a proton motive force in M. barkeri (9). Moreover, in vitro oxidation of CO to CO 2 and H 2 can be reconstituted by using purified Ech, ferredoxin, and acetyl-CoA decarbonylase͞ synthetase (ACDS) (8). A second possible role for Ech involves the first step of methanogenesis from H 2 ͞CO 2 , the reduction of CO 2 to formyl-methanofuran (CHO-MFR).…”

“…Cell suspension experiments have provided evidence that reduction of CO 2 to CHO-MFR is driven by an electrochemical proton or sodium ion gradient (11)(12)(13). Ech and the related hydrogenases Eha and Ehb from Methanothermobacter species have been proposed to function as ion pumps in this reaction providing the low potential reducing equivalents necessary for the reduction of CO 2 to CHO-MFR (1,8). A third possible function of Ech involves the biosynthesis of acetate and pyruvate.…”