title: A1Ao-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 sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(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(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was 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(1)A(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(1)A(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 cyanide m-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 M1 A(1)A(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 low pH and low levels of sodium ions.
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.
authors.forename: Duncan G G
authors.initials: DG
authors.lastname: McMillan
authors.forename: Scott A
authors.initials: SA
authors.lastname: Ferguson
authors.forename: Debjit
authors.initials: D
authors.lastname: Dey
authors.forename: Katja
authors.initials: K
authors.lastname: Schröder
authors.forename: Htin Lin
authors.initials: HL
authors.lastname: Aung
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carbone
authors.forename: Graeme T
authors.initials: GT
authors.lastname: Attwood
authors.forename: Ron S
authors.initials: RS
authors.lastname: Ronimus
authors.forename: Thomas
authors.initials: T
authors.lastname: Meier
authors.forename: Peter H
authors.initials: PH
authors.lastname: Janssen
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000235:genetics; D002414:Cations, Monovalent / Q000378:metabolism; D041581:Methanobrevibacter / Q000201:enzymology* / Q000235:genetics; D008985:Monensin / Q000494:pharmacology; D009876:Operon / Q000502:physiology; D017433:Protein Structure, Secondary; D061209:Proton Ionophores / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D061210:Sodium Ionophores / Q000494:pharmacology
chemical_list: D002414:Cations, Monovalent; D061209:Proton Ionophores; D061210:Sodium Ionophores; D000255:Adenosine Triphosphate; D008985:Monensin; D012964:Sodium; D006180:Proton-Translocating ATPases