Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1.

The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is initially rapid and decreases within about 1 min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of MgATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the enzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme in excess of 1, the initial ATPase activity was low and reached the steady state after about 30 s. Free Mg2+ in the assay mix also inhibited steady-state ATP hydrolysis by all forms of the enzyme. The results are consistent with a model in which two Mg2+ bind cooperatively, probably to the dissociable nucleotide-binding sites on CF1-epsilon. Thus, four different nucleotide-binding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the site to be involved in catalysis.

Subunit movement during catalysis by F1-F0-ATP synthases.

The catalytic portion (F1) of ATP synthases have the subunit composition alpha 3, beta 3, gamma, delta, epsilon. This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual alpha beta pairs with the small subunit must change as binding sites properties alternate. A rotation of the gamma subunit within the alpha 3 beta 3 hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete gamma subunit during ATP hydrolysis is not mandatory for activity. The gamma subunit of chloroplast F1 may be cleaved into three large fragments that remain bound to F1. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of alpha 3 beta 3 have been shown to have significant ATPase activity in the absence of gamma. Mg2+ATP affects the interaction of gamma with the different beta subunits, and induces other changes in F1, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF1 by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.

Differences between two tight ADP binding sites of the chloroplast coupling factor 1 and their effects on ATPase activity.

Purified chloroplast ATP synthase (CF1) contains 1.2-2 mol of tightly bound ADP/mol of enzyme that resists removal by gel filtration or dialysis. CF1 was depleted of its endogenous nucleotide by treatment with alkaline phosphatase. Tightly bound nucleotide was demonstrated not to have an essential structural role. CF1 depleted of endogenous nucleotide retains its ability to catalyze Ca2+- and Mg2+-dependent ATPase activity and is not more sensitive to cold inactivation than untreated CF1. 2'(3')-O-Trinitrophenyladenosine 5'-diphosphate (TNP-ADP) binds tightly to two sites on nucleotide-depleted CF1, binding to either site at a faster rate than that of exchange of bound nucleotide for medium nucleotide. The nucleotide-depleted enzyme binds about one additional mol of TNP-ADP/mol of CF1, indicating that there is a tight TNP-ADP binding site that does not exchange readily with medium nucleotide. It is MgADP in this nonexchanging site, not the easily exchanging ADP binding site, that is responsible for the MgADP-induced inhibition of the ATPase activity. The rate of exchange of tightly bound ADP from CF1 matches the rate at which the Mg2+ATPase activity of CF1 is activated but is not itself responsible for the activation.

Two tight binding sites for ADP and their interactions during nucleotide exchange in chloroplast coupling factor 1.

Chloroplast coupling factor 1 (CF1) deficient in its epsilon subunit was loaded with 2'(3')-O-trinitrophenyl-ADP (TNP-ADP), and the release of tightly bound TNP-ADP was followed as a decrease in fluorescence. TNP-ADP could be exchanged for medium ADP, ATP, MgADP, and MgATP. The preferred substrate for exchange was MgADP, particularly in the presence of P(i). One nucleotide binding site contained ADP which was not displaced during TNP-ADP loading. When Mg2+ was bound at this site, complete exchange of bound TNP-ADP for medium nucleotide was prevented. This tightly bound MgADP was removed by incubation of the enzyme with EDTA. Tightly bound TNP-ADP was removed by high concentrations of sulfite, sulfate, or P(i) in the absence of medium nucleotide and free Mg2+, regardless of the bound Mg2+ content of the enzyme. Sulfite and P(i), in the presence of medium nucleotide and Mg2+, enabled complete exchange of tightly bound TNP-ADP. The combination of Mg2+ and sulfite, or Mg2+ and P(i), caused exchange of tightly bound ADP from two different sites. These results suggest that both sites exchange when the enzyme is fully active, and that at least three sites are likely to participate in catalysis.