Abstract
Rnf complexes are redox-driven ion pumps identified in diverse species from the domains Bacteria and Archaea, biochemical characterizations of which are reported for two species from the domain Bacteria. Here, we present characterizations of the redox-active subunits RnfG and RnfB from the Rnf complex of Methanosarcina acetivorans, an acetate-utilizing methane-producing species from the domain Archaea. The purified RnfG subunit produced in Escherichia coli fluoresced in SDS-PAGE gels under UV illumination and showed a UV-visible spectrum typical of flavoproteins. The Thr166Gly variant of RnfG was colorless and failed to fluoresce under UV illumination confirming a role for Thr166 in binding FMN. Redox titration of holo-RnfG revealed a midpoint potential of −129 mV for FMN with n = 2. The overproduced RnfG was primarily localized to the membrane of E. coli and the sequence contained a transmembrane helix. A topological analysis combining reporter protein fusion and computer predictions indicated that the C-terminal domain containing FMN is located on the outer aspect of the cytoplasmic membrane. The purified RnfB subunit produced in E. coli showed a UV-visible spectrum typical of iron-sulfur proteins. The EPR spectra of reduced RnfB featured a broad spectral shape with g values (2.06, 1.94, 1.90, 1.88) characteristic of magnetically coupled 3Fe-4S and 4Fe-4S clusters in close agreement with the iron and acid-labile sulfur content. The ferredoxin specific to the aceticlastic pathway served as an electron donor to RnfB suggesting this subunit is the entry point of electrons to the Rnf complex. The results advance an understanding of the organization and biochemical properties of the Rnf complex and lay a foundation for further understanding the overall mechanism in the pathway of methane formation from acetate.
Highlights
Acetate is the major source of biological methane in nature
RnfG The His-tagged RnfG subunit was overproduced in E. coli and purified to initiate investigations into electron transfer functions of this eight-subunit Rnf complex from the domain Archaea
Since alkaline phosphatase is only active in the periplasm of E. coli, the results indicate that the location of the C-terminus adjacent to the FMN binding site is at the outer aspect of the cytoplasmic membrane in M. acetivorans consistent with that predicted by the topology algorithms
Summary
Acetate is the major source of biological methane in nature. Only two genera (Methanosarcina and Methanosaeta) of acetateutilizing methane-producing microbes are known of which Methanosarcina species have been researched to the greatest extent. The pathway for conversion of the methyl group of acetate to methane is well documented [1], less is understood concerning electron transport recently reviewed [2]. The great majority of acetate-utilizing Methanosarcina species are unable to metabolize H2 [3] of which Methanosarcina acetivorans is a model. Exceptions to this majority are Methanosarcina barkeri and Methanosarcina mazei which are dependent on the production and consumption of H2 for electron transport coupled to generation of a proton gradient driving ATP synthesis [2]. Methylreductase (Mcr) catalyzes reduction of the methyl group of CH3-S-CoM to methane with electrons donated by the sulfur atoms of coenzyme B (HS-CoB) and CH3S-CoM. The two electrons required for this reduction are derived from oxidation of the carbonyl group of acetate catalyzed by Cdh for which Fd is the electron acceptor
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