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 α-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 ...