Topological and functional relationship of subunits F1-gamma and F0I-PVP(b) in the mitochondrial H+-ATP synthase.
Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-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 F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-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 F0, diamide also induces cross-linking of OSCP with another subunit of F0, 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 F0I-PVP(b) and F1-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 Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-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.
Gaballo
A
A
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Zanotti
F
F
Solimeo
A
A
Papa
S
S
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology*; D017494:Proton Pumps / Q000737:chemistry / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000187:drug effects / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000378:metabolism
D003432:Cross-Linking Reagents; D004220:Disulfides; D017494:Proton Pumps; D013439:Sulfhydryl Reagents; D003958:Diamide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases