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Mechanistic analysis of electron transfer in cytochrome b₅

University of British Columbia

Cytochrome b₅ has a central role in many aspects of eukaryotic cellular metabolism. Its specific function is to transfer electrons from a NADH-fiavoprotein reductase to a variety of physiologically important oxidases. Since the late 1950’s, the chemical and physical properties of cytochrome b₅ have been investigated extensively. Despite the importance of this protein in cellular metabolism and the availablilty of detailed structural information, relatively little attention has been directed toward characterization of the mechanism by which it changes oxidation state. The present work reports a characterization of the oxidation-reduction equilibrium and the reduction kinetics of cytochrome b₅ and two heme-substituted derivauves. Comparison of the functional properties of dimethyl ester heme-substituted cytochrome b₅ with those of the native protein reveals that esterification of the heme propionates increases the reduction potential of the protein by 60 mV and significantly alters the electrostatics of the reduction kinetics with Fe(EDTA)²⁻ as reductant These results support a role for one of the heme propionates in stabilizing the oxidized form of the protein and provide direct evidence for electron transfer at the surface of the protein along the partially exposed heme edge. The functional properties of deuteroheme-substituted cytochrome b₅ are shown to be very similar to those of the nadve protein. Consequendy, this derivative has been used as an optical probe for studying the electron transfer self-exchange rate of cytochrome bi in kinetic analysis of the reaction between the nadve and deuteroheme-subsdtuted proteins. The second-order rate constant for this reaction after correcdon to a driving force of zero is 3.8 x 10²: M⁻¹ s⁻¹ (μ= 0.1 M, pH 7 (phosphate), 25 °C) and represents a prediction of the true self-exchange rate of the native protein. The relatively low magnitude of this rate is attributed to short-range electrostadc effects arising from the heme propionates at the site of electron transfer along the heme edge. This heme-substitution strategy appears to be a generally useful technique for studying self-exchange rates of other hemeproteins with non-covalently bound heme groups.

Text

2010-06-13

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http://hdl.handle.net/2429/25674

Biochemistry and Molecular Biology

University of British Columbia

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