Mapping of the pyrophosphate binding sites of beef heart mitochondrial F1-ATPase by photolabelling with azidonitrophenyl [alpha-32P]pyrophosphate.

4-Azido-2-nitrophenyl [alpha-32P]pyrophosphate (azido-[alpha-32P]PPi) mimics ADP and PPi by some of its binding properties when assayed in the absence of photoirradiation with mitochondrial F1-ATPase. Upon photoirradiation, both alpha- and beta-subunits of F1-ATPase were covalently labelled. Following chemical and enzymatic cleavages of each of the two photolabelled subunits, peptides containing the covalently bound radioactivity were separated by HPLC and identified by amino acid sequencing. Bound azido-[alpha-32P]PPi was found to be concentrated in two distant sequences of the alpha-subunit, namely Asp194-Thr221 and Lys386-Met437, and in a single sequence of the beta-subunit Glu294-Met358 with most of the photoprobe bound to beta-Tyr-311 and beta-Tyr-345. These results are discussed in terms of a model in which the pyrophosphate binding sites of F1 are located in regions of the alpha- and beta-subunits exposed at the interface between the two subunits and correspond to non-catalytic and catalytic adenine nucleotide binding sites, respectively.