The structure and function of the energy-transducing cytochrome b6f complex and cytochrome b-559 intrinsic to the photosystem II reaction center will be studied. The topography of the cytochrome b6 and cytochrome f polypeptide has been determined. (i) The topographical characterization of the cytochrome b6f complex will be completed by extending these studies to the Rieske iron-sulfur protein and subunit IV. The orientation in the membrane will be determined using protease sensitivity of polar epitopes for peptide-directed antibodies, and immunogold labeling of thylakoid vesicles of different sidedness. (ii) Crystallization studies will be continued on the components of the b6f complex, cytochrome f that has been crystallized, co-crystallization of cytochrome f- plastocyanin, crystallization of the Rieske iron-sulfur protein, and of the cytochrome b6f complex using a detergent-solubilized monodisperse preparation of pure dimeric complex. (iii) The function of heme bn of cytochrome b6 in the trans-membrane photosystem I cyclic phosphorylation pathway and as a branch point in the electron transport chain connecting with electron donors on the n-side of the membrane will be assayed by measuring the dependence of ATP synthesis by a single light flash on the state of reduction of heme bn, as well as the dependence of phosphorylation on the inhibitor NQNO whose presence uniquely increases the amplitude of cytochrome b6 reduction by a flash. (iv) Inhibitor-resistant mutants to the inhibitor NQNO will be isolated from the cyanobacteria, Synechococcus sp. PCC 7002 and 7942, and their loci determined by sequencing petD and petB genes. (v) Site-directed mutagenesis of the Arg-86 and Thr-187 residues would address (a) the role of a unique charged residue located near heme bp and (b) the effect of an extra residue inserted between the two His residues in helix IV of cytochrome b6 on its spectral and redox properties and interheme electron transfer rate. (vi) The role of the cytochrome b-559 psbE gene product in binding manganese or calcium essential for oxygen evolution on the lumen (p-) side of the membrane will be tested by inserting a stop codon after the trans-membrane hydrophobic domain. The requirement for the b-559 psbF gene product will be tested by inserting a stop early (G1n-3) in its gene. (vii) A role for cytochrome b-559 as an alternative donor to the PSII reaction center in photoinhibition and stress response, and (viii) the role of thylakoid cytochromes in the pathway of desaturation of the fatty acids of the highly unsaturated thylakoid membrane will be studied.
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