Kinetic effects of the electrochemical proton gradient on plastoquinone reduction at the Qi site of the cytochrome b6f complex.

We have investigated the effects of the light-induced thylakoid transmembrane potential on the turnover of the b(6)f complex in cells of the unicellular green alga Chlamydomonas reinhardtii. The reduction of the potential by either decreasing the light intensity or by adding increasing concentrations of the ionophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) revealed a marked inhibition of the cytochrome b(6) oxidation rate (10-fold) without substantial modifications of cytochrome f oxidation kinetics. Partial recovery of this inhibition could be obtained in the presence of ionophores provided that the membrane potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b(6) oxidation revealed a synergy between the effects of ionophores and the H(2)O-D(2)O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b(6) oxidation at the Qi site and that these reactions are strongly influenced by the light-dependent generation of a transmembrane potential.

Electrochemical and spectral analysis of the long-range interactions between the Qo and Qi sites and the heme prosthetic groups in ubiquinol-cytochrome c oxidoreductase.

The results are presented of an electrochemical and high-resolution spectral analysis of the heme prosthetic groups in the bc1 complex from mouse cells. To study the long-range interactions between the Qo and Qi quinone redox sites and the b heme groups, we analyzed the effects on the proximal and distal b heme groups, and the c1 heme, of inhibitors that tightly and specifically bind to the Qi or Qo redox site. A number of results emerged from these studies. (1) There is inhomogeneous broadening of the b heme alpha band absorption spectra. Furthermore, contrary to the conclusion from low-resolution spectral analysis, the higher energy transition in the split-alpha band spectrum of the bL heme is more intense than the lower energy transition. (2) Inhibitors that bind at the Qi site have significant effects upon the electronic environment of the distal bL heme. Conversely, Qo site inhibitors induced changes in the electronic environment of the distal bH heme. (3) In contrast, inhibitor binding at either site has little effect upon the midpoint potential of the distal heme. (4) Experiments in which both a Qi and a Qo inhibitor are bound at the redox sites indicate that the long-range effects of one inhibitor are not blocked by the second inhibitor; enhanced effects are often observed. (5) In the double-inhibitor titrations involving the Qo inhibitor myxothiazol, there is evidence for two electrochemically and spectrally distinct species of the bL heme group, a phenomenon not observed previously. (6) The high-resolution deconvolutions of alpha band absorption spectra allow an interpretation of these inhibitor-induced changes in terms of homogeneous broadening, inhomogeneous broadening, and changes in x-y degeneracy. The general conclusion from these experiments is that when an inhibitor binds to a quinone redox site of the cytochrome b protein, it produces local conformational changes that, in turn, are transmitted to distal regions of the protein. The ligation of the bH and bL hemes between two parallel transmembrane helices provides a mechanism by which long-distance interactions can be propagated. The lack of long-range effects upon the midpoint potentials of the heme groups suggests, however, that protein conformational changes are unlikely to be a major control mechanism for the transmembrane electron- and proton-transfer steps of the Q cycle.

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity.

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.

Molecular basis for resistance to antimycin and diuron, Q-cycle inhibitors acting at the Qi site in the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae.

The bc1 complex of the mitochondrial respiratory chain transfers electrons from ubiquinol to cytochrome c oxidase. Cytochrome b, a transmembranous protein, is thought to form a transmembrane electron circuit, transferring electrons between two ubiquinone redox sites, (Qi) and (Qo), respectively, near the inner and outer sides of the inner mitochondrial membrane. Antimycin and diuron appear to block cytochrome b oxidation-reduction at one ubiquinone site, presumably Qi. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to antimycin and diuron have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work the mutated amino acid residues from nine antimycin- and three diuron-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. The sequencings were performed by primer extension in the presence of dideoxynucleotides on total mitochondrial RNA preparations using reverse transcriptase. Regions of the cytochrome b protein affecting the inhibitor and putative quinone-binding sites have been defined.