The cytochrome b6-f and b-c1 complex of chloroplasts, mitochondria, and photosynthetic bacteria is an almost universal trans-membrane electron transport and H+ translocating membrane protein complex in energy-transducing membranes. The amino acid sequence and hydropathy functions of the heme binding domain (first 200 residues) of cytochrome b6 is particularly highly conserved. The topography of cytochrome b6 in the membrane will be studied by (i) determining conditions under which the isolated complex is a monomer or dimer; (ii) purifying the cytochrome from the rest of the complex; (iii) incorporating the cytochrome into liposomes and testing the exposure of predicted surface-exposed peptides using (a) antibodies against these peptides, (b) proteolysis by trypsin at a predicted labile site, (c) azido-labaled photoreactive phospholipids, and (d) DCCD as a probe of predicted acidic residues in helix IV. If DCCD is found to react with one of these acidic residues, and it is conserved in cyanobacteria, it would be changed by directed mutagenesis. Crystallization wells are set up of pure active b6-f prepared by a method allowing 5-10 times more yield of complex from the starting material. Studies of the function of cytochrome b6 is thylakoid membranes, reconstituted liposomes, and membranes of cyanobacteria would determine (i) whether heme bp or bn is reduced by a single flash in the presence of NQNO, (ii) whether b6 is reduced by quinol added to liposomes containing b6-f, and whether the Em values of the two hemes can be distinguished in the reconstituted system. (iii) The dependence of b6 flash reduction of the DeltaMuH+ would be determined. Experiments (i) - (iii) should tell whether a Q cycle can be operating. (iv) The questions of whether the msec DeltaPsi that occurs under reducing conditions actually arises from H+ pumping will be tested using an internal pH indicator. (v) The molecular basis of the heterogeneity of the hemes will be tested by deleting Thr-184. (vi) The sites of resistance to inhibitors in the cyanobacterial cyt b6 would be determined by DNA sequencing of selected mutants. These experiments should provide details about the structure of this protein in the membrane, and also determine whether it has a trans-membrane function in non-cyclic or cyclic electron transport.
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