Hybrid Rhodospirillum rubrumF0F1 ATP Synthases Containing Spinach Chloroplast F1 β or α and β Subunits Reveal the Essential Role of the α Subunit in ATP Synthesis and Tentoxin Sensitivity

Abstract

Trace amounts (ϳ5%) of the chloroplast ␣ subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores of Rhodospirillum rubrum with the chloroplast ␤ subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the ␣ subunit in the rebinding of ␤, restoration of catalytic function, and conferral of sensitivity to the chloroplastspecific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast ␣ and ␤ subunits. The treated chromatophores were found to have lost, in addition to most of their ␤ subunits, approximately a third of the ␣ subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum ␣ and chloroplast ␤ subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast ␣ and ␤ subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum ␣ subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast ␣ subunit is required together with the chloroplast ␤ subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the ␤ subunit on sensitivity to tentoxin.The photosynthetic F 0 F 1 ATP synthases found in the thylakoids of chloroplasts and in the cytoplasmic membranes of photosynthetic bacteria couple the movement of protons down an electrochemical proton gradient to the synthesis of ATP during photophosphorylation. The general structure of these ATP synthases is highly conserved, consisting of F 0 , the membrane-spanning proton channel, and F 1 , the peripheral membrane sector, which contains the catalytic sites for reversible ATP synthesis. The F 0 is composed of four different subunits labeled a, b, bЈ, and c in photosynthetic bacteria (1) and I-IV in chloroplasts (2-4). The chloroplast F 0 subunits IV, I, II, and III are analogous to the bacterial a, b, bЈ, and c, respectively, with a probable stoichiometry of a 1 b 1 bЈ 1 c 9 -12 . F 1 from all sources is composed of five different subunits designated ␣ to ⑀ in order of decreasing molecular weight with a stoichiometry ofThe x-ray crystal structure of bovine heart mitochondrial F 1 (MF 1 ) 1 at 2.8-Å resolution (5) defined the three-dimensional structures of alternating ␣ and ␤ subunits as forming a closed hexamer having a portion of the ␥ subunit embedded in its central cavity. Among the nucleotide binding sites, which are located one at each of the six ␣/␤ interfaces, the three catalytic sites, located predominantly on ␤ subunits, were found to exist in three different conformational states. This asymmetric feature is compatible with the binding change mechanism, which...

Structure of spinach chloroplast F 1 -ATPase complexed with the phytopathogenic inhibitor tentoxin

“…Furthermore, the close distance of the side chain NH group of ␣Arg-297 and the main chain carbonyl of L-MeAla-1 (3.2 Å) suggests additional hydrogen bonding between the inhibitor and the ␣-subunit, although this contact was not indicated by HBPLUS (28). The importance of correct hydrogen bonding for the positioning of the inhibitor in the binding cleft is supported by biochemical and mutagenesis studies that demonstrated that tentoxin binding fails in ␤Asp-833Glu mutants or in species that naturally contain glutamate in the position of the essential ␤Asp-83 (7,8). Tentoxin resistance might simply result from the changed orientation of the carboxyl group in the binding cleft, which affects the correct hydrogen bonding and the alignment of the inhibitor.…”

“…Analysis of tentoxin-sensitive and -resistant Nicotiana species indicated that residue ␤Asp-83 located at the interface of the N-terminal ␤-barrel domains in the ␣-and ␤-subunits is crucial for tentoxin binding and͞or sensitivity (7). Substitution of this residue by glutamate, alanine, or leucine caused tentoxin resistance (8), whereas substitution of the native glutamate in the tentoxin-resistant Chlamydomonas reinhardtii F 1 -ATPase by aspartate induced tentoxin sensitivity (9). On the other hand, tentoxin-resistant F 1 -ATPases from thermophilic Bacillus PS3 and from E. coli (10) contain aspartate in the equivalent position of the ␤-subunit as well.…”

Role of the ATP Synthase α-Subunit in Conferring Sensitivity to Tentoxin

“…An additional distinction has been made based on previous studies which have examined tentoxin sensitivity of hybrid enzymes containing the subunit from tentoxin-sensitive chloroplasts. The F 1 enzymes which are normally tentoxin resistant either became tentoxin sensitive (SC) or remained insensitive to tentoxin (RC) when the CF 1 was present (19,21,23). Heavy lines have been drawn above two segments of residues which were used to create chimeric R subunits.…”

“…CF 1 lacking the δ and subunit, R C 3 C 3 γ C , was reconstituted from native R and subunits and recombinant, refolded γ subunit as previously described (22,25). C (26), D83L (23), and γ C (25) were expressed as inclusion bodies in E. coli BL21(DE3)/pLysS cells grown to steady state at 37°C. ATP (grade II) and tentoxin were purchased from Sigma.…”

“…We recently described a method of reconstituting C subunits along with either R R or R C subunits to chromatophores from the photosynthetic bacterium Rhodospirillum rubrum which had been partially depleted of both their R and subunits by LiCl treatment (23). This allowed us to examine the effects of several C mutations on tentoxin sensitivity in the presence of either the bacterial or the chloroplast R subunits.…”