Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Abstract

In F 1 F 0 -ATP synthase, the subunit b 2 ␦ complex comprises the peripheral stator bound to subunit a in F 0 and to the ␣ 3 ␤ 3 hexamer of F 1 . During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F 0 subunits to the binding of F 1 within the assembled holoenzyme, the isolated subunit b dimer, ab 2 subcomplex, and fully assembled F 0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys 64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F 1 (at ␥Cys 106 ) and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F 1 dissociation constants of 2.7 nM for the binding of F 0 and 9 -10 nM for both the ab 2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F 0 contribute to F 1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F 1 to F 0 , whereas subunit a does not contribute significantly.

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

“…It is believed to function as a stator preventing the a 3 b 3 subunits from rotating together with c and e. Studies on the F 1 F 0 ATP synthase from E. coli have revealed the presence of two copies of subunit b forming the second stalk [10,11]. The roles and properties of subunit b within the F 1 F 0 ATP synthase have been investigated [12][13][14][15][16]. Subunit b may serve as an elastic element and store energy during rotational catalysis, but also more active roles in coupling have been suggested [2,9,17,18].…”

Role of the Asymmetry of the Homodimeric b2 Stator Stalk in the Interaction with the F1 Sector of Escherichia coli ATP Synthase

“…The K d value for the interaction of the dimeric form of b with F 1 has been estimated to be in the nanomolar range (21,40,41). Here, using the competitive F 1 -binding assay, we found that both the 79C ϫ 83C and 83C ϫ 90C disulfidelinked heterodimers competed as well as wild-type soluble b but that homodimers competed significantly less well, indicating lower affinity.…”

Targeting cytochrome C oxidase in mitochondria with Pt(II)-porphyrins for photodynamic therapy

“…Two possibilities were considered: resonance energy transfer (RET) to non-fluorescent cytochromes in COX as RET acceptors or reversible electron transfer / triplet state quenching from COX. Fluorescence resonance energy transfer (FRET) to unravel association of proteins [29][30][31] or specific binding of ligands 32 , or to monitor conformational changes like in the rotary biological nanomotor F o F 1 -ATP synthase on the single-molecule level [33][34][35] are most successful with an fluorescent acceptor for ratiometric data analysis. If the acceptor is a quencher only, donor lifetime changes can be used to indicate the existence of FRET.…”