A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions

Duncan G.G Mcmillan,Scott A Ferguson,Debjit Dey,Katja Schröder,Htin Lin Aung,Vincenzo Carbone,Graeme T Attwood,Ron S Ronimus,Thomas Meier,Peter H Janssen,Gregory M Cook

doi:10.1074/jbc.m111.281675

Duncan G.G Mcmillan, Scott A Ferguson + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m111.281675

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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.

Highlights

An enigma in the bioenergetics of methanogens is how the generation of proton and sodium gradients are used to synthesize ATP
Growth of M. ruminantium M1 on H2 and CO2 Is Dependent on the Membrane Potential and a Chemical Gradient of Sodium Ions—M. ruminantium M1 is found in ruminant animals fed a wide variety of diets [30] and grows with H2 plus CO2 and formate, producing methane [31]
To study the role of different electrochemical gradients in the growth of M1, we tested the effect of various uncouplers, ionophores, and ATP synthase inhibitors on the growth of M. ruminantium M1

Summary

Background

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 A1Ao-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A1Ao-ATP synthase (MbbrA1Ao) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Microorganisms have been shown to harbor a number of membrane-bound ATP synthases that are used to synthesize ATP via a proton or sodium ion gradient These enzymes can be divided into three distinct classes as follows: F-type (F1Fo)-ATPases, A-type archaeal (A1Ao)-ATPases, and V-type (V1Vo)ATPases.

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