Regulation of .DELTA.~.mu.H + -coupled ATP synthesis and hydrolysis: role of divalent cations and of the F0F1-.beta. subunit

Abstract

The divalent cation specificity of ATP-linked reactions catalyzed by the H+-translocating F0F! ATP synthase-ATPase complex has been followed in the Rhodospirillum rubrum chromatophore bound complex. In the presence of Mg2+ and Mn2+ the complex catalyzes ATP synthesis and hydrolysis as well as ATP-driven H+ translocation, but in the presence of Ca2+ it catalyzes only ATP hydrolysis, which is not coupled to H+ tanslocation. The inability of Ca2+ to maintain the coupling process is not due to opening of a proton leak in its presence nor to any release of p! from the membrane, because (a) an identical light-induced H+ translocation is observed in the absence or presence of Ca-ATP and (b) the Ca-ATPase, as well as the Mg-and Mn-ATPase activities, is blocked by specific F0 inhibitors. These results indicate that the divalent cations play an important role in the regulation of H+-coupled ATP synthesis and hydrolysis by the F0Fj complex. Further tests suggest that their site of action is located on the Fr/3 subunit. The isolated ß subunit of the R. rubrum FqF, has been reported to contain two nucleotide binding sites, a Mg-independent and a Mg-dependent site [Gromet-Elhanan, Z., & Khananshvili, D. (1984) Biochemistry 23, 1022-1028]. Addition of Mn2+ also enables the binding of 2 mol of ATP/mol of this isolated ß subunit. But under identical conditions, Ca2+ does not enable ATP binding to this cation-dependent site and inhibits its binding in the presence of Mg2+ or Mn2+. In light of these results we propose that the binding of Mg-ATP or -ATP, but not of Ca-ATP, to the R. rubrum ¥3-ß subunit forms the trigger that opens the pathway for H+ translocation through the FqF] complex during catalysis.Electron-transport coupled ATP synthesis in respiratory and photosynthetic organisms is catalyzed by a reversible prot Supported in part by grants from the Minerva Foundation, Munich, West Germany, and from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. ton-translocating ATP synthase-ATPase complex that is composed of two distinct structures (Senior & Wise, 1983; Merchant & Selman, 1985): a readily solubilized catalytic ATPase sector, Fb and an intrinsic membrane sector that is involved in proton fluxes, F0. The F,-ATPase from many different sources has a 3ß3• ( subunit structure and contains 0006-2960/89/0428-3645S01.50/0 © 1989 American Chemical Society

Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

“…The complex fully retained the unique properties of its parent ATPases, which include sensitivity to inhibition by free magnesium (16,17), and a higher CaATPase than MgATPase activity (Table I), both being modulated by ␥ C oxidation and reduction (19,27). Single-molecule studies of extracted native CF 1 (lacking a ␦ subunit) have already provided some evidence for its ␥ subunit rotation (23).…”

“…A second unique feature of ATP synthases of both chloroplasts (16) and chromatophores (17) is their high sensitivity to inhibition by excess free Mg 2ϩ ions, which results in a drastic reduction of their MgATPase activities at a Mg/ATP ratio exceeding 0.5. Another very interesting phenomenon, found in both chloroplasts (18) and chromatophores (17), is the decoupling of Ca 2ϩ -induced ATP hydrolysis from proton translocation, although their membranes do not become leaky to protons. Both systems also show no Ca 2ϩ -dependent ATP synthesis.…”

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

“…C Complexes-An additional and more general tight regulation of the MgATPase activity of both chloroplasts and chromatophores is their sensitivity to inhibition by excess free Mg 2ϩ ions (22,23). Both isolated hybrid complexes showed the same optimal dependence on MgCl 2 concentrations as the native RrF 1 , reaching maximal levels at a Mg/ATP ratio of 0.5 ( Fig.…”

“…CaATPase, unlike the MgATPase, appears only in the AlF x -stabilized, closed ␣ C the catalytic nucleotide-binding site in presence of Ca 2ϩ has no clear catalytic cooperativity and is decoupled from any proton translocation. In a recent study on ␥ rotation in a genetically engineered EcF 1 -␣ 3 ␤ 3 ␥ containing an uncoupled mutation of ␥Met 23 to Lys (18), the mutant ␥ was found to rotate rather similarly to the WT ␥. This unexpected capacity of the uncoupled ␥ subunit to rotate was explained by suggesting that its defective coupling might be after ␥ rotation, possibly in the interactions between the F 1 and F 0 sectors (46).…”

“…Plant chloroplasts and bacterial chromatophores have a number of such regulatory pathways, which operate in their membranebound F 0 F 1 -as well as the solubilized F 1 -ATPases. Both chloroplasts (22) and chromatophores (23) show a high sensitivity to inhibition by excess free Mg 2ϩ ions, which results in a drastic decrease of their MgATPase activities at Mg/ATP ratios above 0.5. A unique chloroplast regulatory system, termed thiol modulation, involves reduction-oxidation of a disulfide bond (24) formed between Cys 199 and Cys 205 in a region of its ␥ C subunit that does not appear in any other F 1 -␥ subunits (25).…”