Stoichiometry and deletion analyses of subunits in the heterotrimeric F‐ATP synthase c ring from the acetogenic bacterium Acetobacterium woodii

Abstract

The ion‐translocating c ring of the Na+ F1Fo ATP synthase of the anaerobic bacterium Acetobacterium woodii is the first heteromeric c ring found in nature that contains one V‐ (c1) and two F‐type‐like c subunits (c2/c3), the latter of identical amino acid sequence. To address whether they are of equal or different importance for function, they were deleted in combination or individually. Deletion of c1 was compensated by incorporation of two c2/c3 subunits but the enzyme was unstable and largely impaired in Na+ transport. Deletion of c2 was compensated by incorporation of c3 but also led to a reduction of Na+ transport. Deletion of c3 had no effect. In contrast, deletion of both c2 and c3 led to a complete loss of ATPase activity at the cytoplasmic membrane. Mass spectrometric analysis of c2+1 Ala and c2+2 Ala variants revealed a copy number of 8 : 1 for c2/c3 which is consistent with the biochemical characteristics of the variants. These data indicate a role of c1 in assembly and a function of c2 as the predominant c ring constituent.

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD

“…As demonstrated for other oligomeric proteins like the c -subunits of thermophilic 31 and archaeal ATP synthases, 32,33 only autoclaving of these proteins enabled a complete denaturation of the proteins. Therefore, PanD WT as well as PanD H21R and PanD 127TRASC131 were autoclaved in the presence of 1 mM of DTT and subsequently applied onto an SDS gel.…”

The structural features of Acetobacterium woodii F‐ATP synthase reveal the importance of the unique subunit γ‐loop in Na+ translocation and ATP synthesis

“…Together, they form a ring of 10 c subunits with a c 1 : c 2/3 stoichiometry of 1 : 9, as confirmed by structural studies , and a c 1 : c 2 : c 3 stoichiometry of 1 : 8.3 : 0.7 as revealed by laser‐induced liquid beam ion desorption mass spectrometry . Deletion of atpE 1 or aptE 2 significantly diminished ATPase activity and Na + translocation in the Δ c 1 or Δ c 2 deleted mutant enzyme . In comparison, deletion of subunit c 3 did not alter ATP hydrolysis activity and had a minor effect on Na + transport.…”

“…Previous studies on the deletion of the c 2 subunit led to a mutant variant with an ATP hydrolysis‐ and Na + transport activity of about 35% and 40%, respectively, compared to the WT enzyme . The A. woodii F‐ATP synthase model reveals that the residues K60, S61, G62, N63, K198, and K200 of the rotary subunit γ are in proximity to the c 2 subunit (Fig.…”

“…While c 2 and c 3 are two identical bacterial F O -like c subunits that form a hairpin in the membrane with two transmembrane helices, c 1 consists of a double-hairpin structure that resembles the c-subunit of eukaryotic V 1 V O -ATPases [8]. Together, they form a ring of 10 c subunits with a c 1 : c 2/3 stoichiometry of 1 : 9, as confirmed by structural studies [4,5], and a c 1 : c 2 : c 3 stoichiometry of 1 : 8.3 : 0.7 as revealed by laser-induced liquid beam ion desorption mass spectrometry [9]. Deletion of atpE 1 or aptE 2 significantly diminished ATPase activity and Na + translocation in the Dc 1 or Dc 2 deleted mutant enzyme [9].…”

“…C). This implies that subunit c 1 represents a kind of bearing for the A. woodii subunit ε, and would in part explain the reduced amount of subunit ε observed in the Δ c 1 ‐variant as well as its diminished enzyme activity and Na + translocation .…”