Identification of functionally important regions of adrenal and duodenal cytochromes b561

Adrenal and duodenal cytochromes b561 (cyt b561 and Dcytb, respectively) are transmembrane proteins sharing four conserved histidines that provide axial ligands to the low potential (bL) and high potential (bH) hemes; both proteins shuttle electrons from ascorbate. Spectrophotometric analyses of recombinant cyt b561 with Ala-scanning mutations in extra-membrane segment 3 (EM3, residues 70-85) and cyt b561 chimeras carrying EM3, EM5, or EM7 from Dcytb were used to indentify structural features affecting the interaction with ascorbate. For cyt b561 the midpoint ascorbate concentrations (in μM) were 5.2 ± 0.4 (bH) and 318 ± 50 (bL). Significant changes in the bH midpoint were found for R74A (11.2 ±1.3), R77A (13.8 ± 1.8), and R82A (2.1 ± 0.8); the bL midpoint was altered in R74A (1022 ± 130), R77A (705 ± 179), and T83A (182 ± 39). The cyt b561 chimera with EM5 from Dcytb showed large changes in both ascorbate midpoints (28 ± 3; 873 ± 50). These results identify several functionally relevant residues in EM3 o...