Unmasking cooperativity of oxidative phosphorylation by a new α‐phosphate acylated ADP‐analog

Aromatic 3’esters of ADP are surprisingly strong inhibitors of oxidative and photophosphorylation, whereas their inhibitory activity on uncoupled membrane-bound or isolated F,-ATPase is >200-times lower [ 1,2]. Therefore they have been tentatively assigned conformation-specific probes of the catalytic site, assuming that only in ‘energized’ membranebound coupling factors the catalytic site is ‘open’ for these analogs [ 1,3]. Accidently it was observed, that synthesis of 3’esters can be directed to yield larger amounts of side-product acylated at the a-phosphate of ADP instead of acylation of ribose. This is shown here for the fluorescent derivatives 3’-0(5-dimethylamino-naphthoyl-l)-ADP and the respective cy-P-acylderivative. Unexpectedly, also the ol-P-acylated ADPanalog strongly inhibits oxidative phosphorylation, and moreover unmasks cooperativity of this fundamental process of biological energy conversion. Results of kinetic studies suggest, that the mechanism involves two interdependent nucleotide binding sites.