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

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b561 have been proposed to be involved in this cytochrome's 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 70-85) 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 (=94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the a-band line shape and amplitude during reduction of the high- and low-potential heme centers (bH and bL, respectively) and the midpoint ascorbate concentrations for the bH and bL transitions (CH and CL, respectively). Y73A and K85A markedly narrowed the bH a-band peak; other mutants had weaker effects or no effect on bH or bL spectra. Relative changes in CH for the mutants were larger than changes in CL, with 1.5-2.9-fold increases in CH for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional bH and bL 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 bH heme center, and on formation of the native bH-bL 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 bH near the cytoplasmic side of the membrane. [ABSTRACT FROM AUTHOR]