Site-directed mutants Y317C, Y317E, Y317F, Y317G, and Y317K were made to the catch-loop tyrosine on the β subunit of the chloroplast F1-ATPase in Chlamydomonas. EPR spectra of VO2+-ATP bound to site 3 of CF1 from wild type and mutants were obtained. Every mutant changed the 51V hyperfine parameters of the VO2+ bound at this site in the catalytically active conformation of the enzyme but had no effect on these parameters in the form that predominates when the enzyme activity is latent. These results indicate that this residue is a ligand to the metal of the Mg2+-nucleotide complex that binds to the empty catalytic site. The mutations also decreased the kcat of the ATPase activity to a much greater extent than kcat/KM. Thus, these mutations limit the rate of product (Mg2+-ADP and phosphate) release in the ATPase direction or, conversely, the initial binding of substrates in the ATP synthesis direction. On the basis of these observations, coordination of βY317 by Mg2+-ADP that binds to the empty catalytic site provides a means by which substrate binding could trigger γ subunit rotation and consequent conformation changes of β subunits during ATP synthesis.
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