Cysteine-Reactive Fluorescence Probes of Catalytic Sites of ATP Synthase

We searched for new fluorescent probes of catalytic-site nucleotide binding in F1F0-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F1 with thiol-reactive fluorescent probes. Four suitable mutant/probe combinations were identified. βF410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal changes in response to nucleotide, allowing facile measurement of fluorescence and nucleotide-binding parameters, not only in F1 but also in F1F0. The results are consistent with the presence of three asymmetric catalytic sites of widely different affinities, with similar properties in both enzymes, and revealed a unique probe environment at the high-affinity site 1. βY331C F1 labeled by ABD-F gave a large signal which monitored catalytic site polarity changes that occur along the ATP hydrolysis pathway. Two other mutant/probe combinations with significant nucleotide-responsive signals were βY331C labeled by 5-((((2-iodoacetyl)amino)ethyl)amino)naphthaline-1-sulfonic acid and αF291C labeled by 2-4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid. The signal of the latter responds differentially to nucleoside diphosphate versus triphosphate bound in catalytic sites.