Probing the Differences between Rat Liver Outer Mitochondrial Membrane Cytochrome b5 and Microsomal Cytochromes b5

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• 作者：Adriana Altuve ; Svetlana Silchenko ; Kyung-Hoon Lee ; Krzysztof Kuczera ; Simon Terzyan ; Xuejun Zhang ; David R. Benson ; and Mario Rivera
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 14, 2001
• 年：2001
• 卷：40
• 期：32
• 页码：9469 - 9483
• 全文大小：731K
• 年卷期：v.40,no.32(August 14, 2001)
• ISSN：1520-4995

文摘

Two distinct forms of cytochrome b₅ exist in the rat hepatocyte. One is associated with themembrane of the endoplasmic reticulum (microsomal, or Mc, cyt b₅) while the other is associated withthe outer membrane of liver mitochondria (OM cyt b₅). Rat OM cyt b₅, the only OM cyt b₅ identified sofar, has a significantly more negative reduction potential and is substantially more stable toward chemicaland thermal denaturation than Mc cytochromes b₅. In addition, hemin is kinetically trapped in rat OM cytb₅ but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b₅ to apomyoglobin isobserved at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientationalisomers be achieved under physiologically relevant conditions. These differences are striking given thesimilarity of the respective protein folds. A combined theoretical and experimental study has been conductedin order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b₅. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cytb₅ revealed a conformational change that exposes several internal residues to the aqueous environment.The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reportedMD simulation of bovine Mc cyt b₅ [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restrictingaccess of water to the protein interior. Subsequent comparisons of the protein sequences and structuressuggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36,and Leu-47 might contribute to the inability of rat OM cyt b₅ to adopt the cleft-opened conformation and,hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mccyt b₅ because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe theroles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b₅ with its unusual structural properties,we have replaced them with the corresponding residues in bovine Mc cyt b₅. Hence, the I32L (single),A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b₅ were prepared. The stabilityof these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OMA18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b₅. The decrease in stability of the rat OMprotein correlates with the extent to which the hydrophobic cluster involving the side chains of residues18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triplemutant is confirmed in a 2.0 Å resolution crystal structure of the protein. Disruption of the hydrophobicnetwork also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triplemutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, ¹H NMR spectroscopy andside-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32Lcyt b₅ allowed us to conclude that the nature of residue 32 plays a key role in controlling the relativestability of hemin orientational isomers A and B in rat OM cyt b₅. A similar analysis led to the conclusionthat Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromesb₅.