ATP HYDROLYSIS-DRIVEN STRUCTURAL-CHANGES IN THE GAMMA-SUBUNIT OF ESCHERICHIA-COLI ATPASE MONITORED BY FLUORESCENCE FROM PROBES BOUND AT INTRODUCED CYSTEINE RESIDUES

Four mutants of the Escherichia coli F(1)ATPase, gamma S8-C, gamma T10 6-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent 4-7-(diethylamino)4-methylco umarin-3-yl)-maleimide (CM) under conditions that selectively label th e introduced Cys residue. With each mutant the effect of nucleotide bi nding on the fluorescence of the probe has been monitored. The results obtained with the mutants gamma S8-C and gamma T106-C are similar. In both cases, there was a spectral shift and change in fluorescence int ensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from c atalytic sites, while no change in the fluorescence spectrum was obser ved upon adding ADP. The fluorescence spectral changes obtained with A TP were transient and involved an initial rapid fluorescence enhanceme nt followed by a subsequent fluorescence quenching. The kinetics of th ese ATP-induced fluorescence changes and the kinetics of ATP hydrolysi s as monitored by the rates of ATP binding and of Pi formation were th e same under conditions of unisite catalysis, indicating that the conf ormational changes in the gamma-subunit being measured by the fluoresc ent probe are driven by ATP hydrolysis in catalytic sites. No nucleoti de-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were se en with CM bound at position 286, but these appear to reflect structur al changes due to binding of ADP or ATP in noncatalytic sites. The flu orescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon free E. coli F(1)ATPase, although such en zyme preparations are highly active ATPases. We conclude that the stru ctural changes monitored by the fluorescent probe are a part of the co nformational coupling, whereby catalytic site events are linked to pro ton channeling.