Topological and functional relationship of subunits F1-γ and FoI-PVP(b) in the mitochondrial H+-ATP synthase

Diamide treatment of the FoF1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated F-o reconstituted with F-1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the FoI-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F-1-gamma, both located in the stalk. The FoI-PVP(b) and F-1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that FoI-PVP(b) and F-1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of FoI-PVP(b) and the short alpha-helix of F-1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F-o, diamide also induces cross-linking of OSCP with another subunit of F-o, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between FoI-PVP(b) and F-1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through F-o induces a relative motion and/or a separation of the FoI-PVP(b) subunit and F-1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.