Functional and Structural Roles of Residues in the Third Extramembrane Segment of Adrenal Cytochrome b561

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• 作者：Wen Liu ; Giordano F. Z. da Silva ; Gang Wu ; Graham Palmer ; Ah-Lim Tsai ; Richard J. Kulmacz
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：April 19, 2011
• 年：2011
• 卷：50
• 期：15
• 页码：3149-3160
• 全文大小：1133K
• 年卷期：v.50,no.15(April 19, 2011)
• ISSN：1520-4995

文摘

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b₅₆₁ have been proposed to be involved in this cytochrome鈥檚 interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70鈭?5) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (鈮?4% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the 伪-band line shape and amplitude during reduction of the high- and low-potential heme centers (b_H and b_L, respectively) and the midpoint ascorbate concentrations for the b_H and b_L transitions (C_H and C_L, respectively). Y73A and K85A markedly narrowed the b_H 伪-band peak; other mutants had weaker effects or no effect on b_H or b_L spectra. Relative changes in C_H for the mutants were larger than changes in C_L, with 1.5鈭?.9-fold increases in C_H for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b_H and b_L centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b_H heme center, and on formation of the native b_H鈭?i>b_L unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b_H near the cytoplasmic side of the membrane.