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Interaction of cytochrome b₅ and cytochrome c

University of British Columbia

The interaction and kinetics of electron transfer between cytochrome b₅ and cytochrome c, two well characterised soluble electron transfer proteins, have been investigated by three techniques. First, fluorescence quenching experiments were done with cytochrome b₅ and porphyrin cytochrome c, a fluorescent analogue of cytochrome c. These quenching studies yielded association constants and structural information for the cytochrome b₅ cytochrome c complex. Second, an analysis of the rate of flavin semiquinone reduction of the cytochromes within the cytochrome b₅ cytochrome c complex yielded information about the structure and electrostatics of die complex. Third, the second order rate constant for ferrocytochrome b₅ reduction of ferricytochrome c was determined under a variety of solution conditions by stopped-flow spectroscopy. Particular effort was directed at evaluating the role of the cytochrome bs heme propionates in the interaction and electron transfer between cytochrome b₅ and c through performing each of diese three studies with a derivative of cytochrome b₅ in which die heme propionates had been esterified (referred to as DME-cytochrome b₅). The fluorescence quenching studies on the interaction of cytochrome b₅ and porphyrin cytochrome c and die kinetics of flavin semiquinone reduction of the proteins within the cytochrome b₅ cytochrome c complex provided evidence that esterification of the cytochrome b₅ heme propionates, influences the docking geometry of the two proteins. These findings support the predictions of electrostatic calculations [Mauk, M.R., Mauk, A.G., Weber, P.C. & Matthew, J.B. (1986) Biochemistry, 25, 7085] in two respects. First, esterification of the cytochrome b₅ heme propionates detectably increases the separation between die two heme groups in die cytochrome b₅ cytochrome c complex. Second, die cytochrome c heme group is not as sterically hindered in the DME-cytochiome b₅ cytochrome c complex as in die cytochrome b₅ cytochrome c complex. The stopped-flow studies demonstrate that the bimolecular rate constant for ferrocytochrome b₅ reduction of ferricytochrome c is determined in part by the rate of association of the two proteins. This rate of association is strongly influenced by electrostatic forces. The principal effect of esterification of the cytochrome b₅ heme propionates on the second order rate of electron transfer between cytochromes bs and c is to influence complex formation between these two proteins. The stopped-flow studies further suggest that the reduction potentials of native and DME-cytochromes b₅ are not significantly different when these proteins are bound to cytochrome c. The nature of electron transfer protein-protein interactions and protein-protein electron transfer is discussed with respect to these findings.

Text

2010-10-11

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http://hdl.handle.net/2429/29096

Biochemistry and Molecular Biology

University of British Columbia

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