The a subunit of the A1AO ATP synthase of Methanosarcina mazei Gö1 contains two conserved arginine residues that are crucial for ATP synthesis.
Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. [Formula: see text] as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed.
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Carolin
C
Gloger
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Anna-Katharina
AK
Born
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Martin
M
Antosch
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Electronic address: vmueller@bio.uni-frankfurt.de.
Volker
V
Müller
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005816:Genetic Complementation Test; D017020:Methanosarcina / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D021122:Protein Subunits; D009243:NAD; D000255:Adenosine Triphosphate; D001120:Arginine; C113203:A1A0 ATPase, Methanosarcina mazei; D006180:Proton-Translocating ATPases
Archaea; Energy conservation; Ion translocation; Methanogens; Site directed mutagenesis