Abstract
A gene (isf) encoding an iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was cloned and sequenced. The gene was located directly upstream of the genes (pta and ack) encoding phosphotransacetylase and acetate kinase and is transcribed in the opposite direction. The amino acid sequence deduced from isf contained a cluster of cysteine residues reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center. The protein was heterologously produced in Escherichia coli and purified to apparent homogeneity. The 29-kDa subunit molecular mass of heterologously produced Isf (determined by SDS-polyacrylamide gel electrophoresis) corresponded to the molecular mass of 30,451 Da calculated from the amino acid composition deduced from isf. Gel filtration estimated a molecular mass of 65 kDa for the native Isf indicating an alpha2 homodimer. The UV-visible absorption spectrum was characteristic of iron-sulfur flavoproteins with maxima at 484, 452, 430, 378, and 280 nm. Analyses identified 2 FMN, 7-8 non-heme iron atoms, and 6-7 acid-labile sulfur atoms per alpha2 homodimer. Comparisons of the deduced Isf sequence with sequences in available protein data bases suggested Isf is a novel iron-sulfur flavoprotein. Western blot analysis indicated the presence of Isf in extracts of acetate-grown M. thermophila. Ferredoxin stimulated the CO-dependant reduction of Isf by the CO dehydrogenase middle dotacetyl-CoA synthase complex that suggested ferredoxin is a physiological electron donor to Isf.
Highlights
Produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in the methanoarchaea
The methyl group is transferred to HS-CoM, and the CH3-S-CoM is reduced with electrons from HS-CoB to release CH4 and the heterodisulfide CoM-S-S-CoB
A novel flavoprotein that contains FMN as the prosthetic group was purified from the methanoarchaeon Methanobacterium thermoautotrophicum where it is proposed to function as an electron carrier in the CO2-reducing pathway [14, 15]
Summary
Produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in the methanoarchaea. We report on a novel FMN-binding iron-sulfur protein (Isf) in acetate-grown M. thermophila. Iron-Sulfur Flavoprotein from M. thermophila the upstream primer was 5Ј-GAGCGATCCATATGAAAATAACAGGA-3Ј that corresponds, in part, to nucleotides 1–15 in Fig. 2 and contains the recognition sequence for NdeI.
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