Abstract
Two membrane‐bound isoforms of cytochrome b~5~ have been identified in mammals, one associated with the outer mitochondrial membrane (OM b~5~) and the other with the endoplasmic reticulum (microsomal, or Mc b~5~). The soluble heme binding domains of OM and Mc b~5~ have highly similar three‐dimensional structures but differ significantly in physical properties, with OM b~5~ exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5‐ns length molecular dynamics simulations for rat Mc b~5~, bovine Mc b~5~, and rat OM b~5~, as well as for two rat OM b~5~ mutants that were anticipated to exhibit properties intermediate between those of rat OM b~5~ and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM^3M^) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM^5M^). Analysis of the structure, fluctuations, and interactions showed that the five b~5~ variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b~5~ and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b~5~, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682–9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469–9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM^5M^ b~5~ verify that stability and dynamic properties of cytochrome b~5~ are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein. © 2006 Wiley Periodicals, Inc. Biopolymers 83:297–312, 2006
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