STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES

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The highly conserved cytochrome b6f complex and the reaction center cytochrome b-559 of oxygenic photosynthesis will be used to study basic aspects of the structure and function of integral membrane cytochromes. Thr proposed studies are based on the following results from previous grant support: (i) the solution of the crystal structure at 2.3A resolution of the active major extrinsic domain of cytochrome f, the first structure at the atomic level of a subunit of the cytochrome bc1 or b6f complexes. Cytochrome f has three unprecedented structural features for a c-type cytochrome of (a) a predominant beta-strand motif, (b) two distinguishable domains, and (c) the N-terminal alpha-amino group of Tyr-1 as the axial sixth heme ligand: the latter result provided specific information about the sequence of events in the translocation of cyt f across the membrane, i.e. that processing must precede completion of heme coordination and final assembly. (ii) The purified b6f complex was characterized as a structural and functional dimer. (iii) The interhelix forces of the b6f complex were found to be relatively weak. (iv) The orientation of the beta-subunit of that heme cross-linked cytochrome b-559 was found to be parallel to that of the alpha, in agreement with (a) the prediction that it is a heterodimer, (b) the cis-positive rule for orientation of membrane proteins, and (c) the calculated contribution of the dipole potential of the alpha and beta helices to its very positive midpoint potential. It is proposed; (I) to use the cytochrome f structure and existing cross- linking information as the basis for 'intelligent' site-directed mutagenesis to (a) determine the position of the docking site(s) for plastocyanin, and (b) to make a set of single histidine surface mutants that will be utilized, after modification wit Ru (bpy)2 adducts, to measure the reorganization energy, gamma, and optimum pathway associated with the intraprotein electron transfer. It is hypothesized that this transfer will have an unusually small gamma because the transfer to the plastocyanin acceptor is isopotential. (c) The consequences for assembly of cyt f and the subunits of the complex will be tested of inhibition of the processing and liberation of the Tyr-1 amino group. (II,a) The homodisperse Mr 230,000 b6f dimer will be used to crystallize this integral membrane protein complex. (b) The function of dimeric b6f complex will be tested in trans-membrane signaling involving the n-side kinase, as will (c) the role of the very highly conserved n-side extrinsic loops of cyt b6 in the docking of peripheral proteins. (iii) The effect of topographical inversion of the cyt b-559 heme will be examined by applying the cis- positive rule and reversing its trans-membrane distribution of positively charged amino acids.