Tight nucleotide binding sites and ATPase activities of theRhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase

Abstract

Solubilized Rhodospirillum rubrum RrF1-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF1 with Mg(2+)-ATP or Mg(2+)-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF1. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF1 has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F1-ATPases have faster koff constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg2+ than Ca(2+)-ATPase activity observed with native RrF1, as with epsilon-depleted, activated CF1. An almost complete conversion of both RrF1 and CF1 from Ca(2+)- to Mg(2+)-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF1 and CF1 divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg2+ ions. The RrF1-ATPase activity is 30-fold more sensitive than CF1 to efrapeptin, and completely resistant to either inhibition or stimulation by the CF1 effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF1 a low-affinity, stimulatory site for tentoxin.

Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits

“…This mutant complex was expressed and purified in large amounts and responded to the ␥ C thiol modulation, but even its DTT-reduced MgATPase activity reached at the most 5 units/mg. Furthermore, no CaATPase activity has been reported in this mutant TF 1 -␣ 3 ␤ 3 ␥ complex, probably because in the native TF 1 , unlike in RrF 1 (Table I) and CF 1 (30), the CaATPase activity is 10-fold lower than its MgATPase activity (43).…”

“…Since azide does not inhibit the CaATPase activity, another inhibitor will be required for such comparative studies. A very suitable candidate is the specific CF 1 effector tentoxin, which at low concentrations inhibits but at high concentrations stimulates both CF 1 Ca-and MgATPase activities (30). RrF 1 is, however, completely resistant to tentoxin.…”

Formation and properties of hybrid photosynthetic F1‐ATPases

“…The hybrid enzyme assembly exhibited several properties distinct from those of the native RrF 1 and CF 1 enzymes and from the earlier tested hybrid assembly [19,33]. The hybrid most closely resembled the complex in that both enzymes hydrolyze MgATP very slowly in the absence of stimulating oxyanions while both have significant rates of Ca‐ATPase activity.…”

“…The activities of both the and enzymes continued to increase in parallel at tentoxin concentrations up to 400 µ m (not shown). As the native R. rubrum F 1 enzyme is neither inhibited nor stimulated by tentoxin [33], the result suggested that the CF 1 β subunit was responsible for conferring tentoxin stimulation upon the enzyme. This surprising result was confirmed by an experiment in which the wild‐type CF 1 β subunit was replaced with a mutant CF 1 β subunit in which the aspartate residue at position 83 had been replaced with leucine.…”

The photosynthetic F1-?3?3 and ?1?1 catalytic core complexes

“…ATPase activity was assayed with 5 mM MgCI2 and 0.2 mM ATE and when added 2 mM azide, 16 mM octylglucoside and 10 mM sulfite. bData modified from Weiss et al (1994). ATPase activity was assayed with 2 mM MgCI2 and 4 mM ATE The concentrations of added effectors were as for RrFt-cq/.31, and when stated low tentoxin, 1/~M and high tentoxin, 100/zM.…”