ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies.
A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit. When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000. When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000. The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit. We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
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Aggeler
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Capaldi
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D001386:Azides; D003432:Cross-Linking Reagents; D008301:Maleimides; C073709:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-6-maleimidohexanamide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium