The subunit b dimer of the FoF1-ATP synthase interaction with F1-ATPase as deduced by site-specific spin-labeling

We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F-1-ATPase. Interaction of b(2) with a delta-depleted F-1 (F-1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b(2) in the presence of F-1 or F-1-delta when compared with the spectra of free b(2) indicate a tight binding interaction between b(2) and F-1. The data suggest that b(2) packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F-1-ATPase as well as to F-1-delta. Subsequent addition of delta to F-1-delta.b(2) complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F-1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.