A 1A o-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions

Abstract

An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodiummotive forces during methane production is used to synthesize ATP by the membrane-bound A 1A o-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A 1A o-ATP synthase (MbbrA 1A o) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantiumM1was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA 1A o was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na +-binding signature made up of identical amino acid residues. The purified MbbrA 1A o was stimulated by sodium ions, and Na + provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanidem-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1A 1A o- ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as lowpHand low levels of sodium ions. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.