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We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.

The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated. Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of epsilon(c) or epsilon(t) to the alpha3beta3gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.

CF0CF1 was isolated from chloroplasts of the cell wall-deficient Chlamydomonas reinhardtii strain cw15. The subunit pattern was analyzed by SDS-gel electrophoresis and the N-terminal amino acid sequences of all nine subunits were determined by microsequencing. The amino acid sequences of subunits alpha, beta, gamma and epsilon match with those derived from the corresponding Chlamydomonas DNA sequences. In variance with the previously assumed N-terminus of beta; however, it was found that the first 11 amino acids are lacking. The subunits delta, I, II, III and IV were identified by comparison with known sequences of homologous polypeptides of higher plant chloroplasts and cyanobacteria, respectively.

Fluorescence quenching of 9-aminoacridine was followed to ascertain the state of light-induced transthylakoidal Δ μ H + at energetic equilibrium with a subsequently added mixture of ATP, ADP and phosphate. In the measured range, the logarithm of relative fluorescence quenching at equilibrium is a linear function of the imposed phosphate potential. The slope of the line is shown by mathematical deduction to be equal to the ATP/H + stoichiometry of the H + -coupled reversible ATPase reaction. Determination of the stoichiometry by this method neither relies on the standard phosphate potential nor on the internal thylakoid volume. The results confirm that during ATP synthesis or hydrolysis 3 H + per ATP are translocated through the CF 0 -CF 1 complex.

We produced atpA and atpB deletion mutants of C. reinhardtii cw15 in order to investigate the role of certain domains of the α and β subunits of the chloroplast ATP synthase (CF 0 CF 1 ) by site-directed mutagenesis. The deletion mutants were obtained by transformation with constructs containing the aadA cassette inserted into the atpA or atpB genes in place of the corresponding open reading frames. Homoplasmic strains were obtained by continued selection under heterotrophic growth conditions in the presence of spectinomycin. The deletion mutants could be complemented by transformation with the wild-type atpA or atpB genes, respectively. In both deletion mutants none of the subunits of CF 1 and of CF 0 were detected by electrophoretic analysis. Since the mother strain cw15 as well as the deletion mutants are cell wall-deficient, we were able to prepare photosynthetically active thylakoids and to study the photosynthetic characteristics of cw15 thylakoids and mutant thylakoids lacking the ATP synthase. Electron transport measurements showed that the PSII and PSI activities were not affected in the deletion mutants. Thylakoids from the deletion mutants were able to maintain a significantly higher light-induced proton gradient than thylakoids from cw15, confirming the absence of a functional proton channel in these deletion mutants. When substrates of photophosphorylation were added, the transmembrane proton gradient in wild-type thylakoids decreased and ATP was formed, while in the deletion mutant thylakoids the proton gradient was not affected and no ATP was formed. The phosphorylation/ Δ pH relationship in C. reinhardtii thylakoids was shifted to lower Δ pH values by reduction with dithiothreitol, indicating that the C. reinhardtii enzyme is also similar to the higher plant chloroplast ATP synthase in this respect.

In this paper the authors emphasise that the proton translocating ATP synthase from thiol-modulated chloroplasts and two cyanobacterial strains has a coupling ratio of 4 protons per ATP synthesised or hydrolysed. This ratio is determined by several thermodynamic studies at equilibrium between phosphate potential (ΔGp) and proton gradient ( Δ μ H + ), and is confirmed by measurement of proton flux during ATP hydrolysis. Ratios lower than 4 H+/ATP that have been published in the past have predominantly been determined with the oxidised chloroplast enzyme. Errors in these measurements will be discussed.

Thylakoid energization by light causes destabilization of CF0CF1 so that the peripheral CF1 sector is more readily detached from the membrane by intermediate concentrations of the chaotropic salt NaSCN. Here we have investigated the correlation between the proton gradient-induced change of CF0CF1 interaction and CF0CF1 activation. The results indicate a close relationship between the two phenomena. The effect is most probably due to reduction of the electrostatic interaction between the two subcomplexes CF0 and CF1 as a consequence of protonations in the interface region.