PROTON SLIP OF THE CHLOROPLAST ATPASE - ITS NUCLEOTIDE DEPENDENCE, ENERGETIC THRESHOLD, AND RELATION TO AN ALTERNATING SITE MECHANISM OF CATALYSIS

The F-ATPase of chloroplasts couples proton flow to ATP synthesis, but is leaky to protons in the absence of nucleotides. This ''proton slip'' can be blocked by small concentrations of ADP or by inhibitors of the channel portion, CF0. We studied charge flow through the ATPase by flash spectrophotometry and analyzed the inhibition of proton slip by nucleotides, phosphate/arsenate, and insufficient proton motive force. The following inhibition constants (at given background concentrations) were observed: ADP, 0.2 muM (0.5 mM P(i)); ADP, 13.4 muM (no P(i)); P(i), 43 muM (1 muM ADP); GDP, 2.5 muM (0.5 mM P(i)); ATP, 2 muM. ADP and P(i) mutually lowered their respective inhibition constants. Phosphate could be replaced by arsenate. Proton slip occurred only if the proton motive force exceeded a certain threshold, similar to that for ATP synthesis. The inhibition of proton slip by ADP and GDP qualified the respective nucleotide binding sites as belonging to the subset of two (or three) potentially catalytic sites out of the total of six. We interpreted the ADP-induced transition between different conduction states of the ATPase from ''slipping'' to ''closed'' to ''coupled'' as a consequence of the alternating site mechanism of catalysis. Where as the proton translocator idles in the absence of nucleotides, the high-affinity binding of the first ADP/P(i) couple to one site clutches proton flow to some (conformational) change that can only be executed after the binding of another ADP/P(i) couple to a second site. From there on these sites alternate in the catalytic cycle. An entropic machine is presented which likewise models proton slip, unisite, and multisite ATP synthesis and hydrolysis.