Identification of functionally important regions of adrenal and duodenal cytochromes b561

Adrenal and duodenal cytochromes b 561 (cyt b 561 and Dcytb, respectively) are transmembrane proteins sharing four conserved histidines that provide axial ligands to the low potential (b L ) and high potential (b H ) hemes; both proteins shuttle electrons from ascorbate. Spectrophotometric analyses of recombinant cyt b 561 with Ala‐scanning mutations in extra‐membrane segment 3 (EM3, residues 70‐85) and cyt b 561 chimeras carrying EM3, EM5, or EM7 from Dcytb were used to indentify structural features affecting the interaction with ascorbate. For cyt b 561 the midpoint ascorbate concentrations (in μM) were 5.2 ± 0.4 (b H ) and 318 ± 50 (b L ). Significant changes in the b H midpoint were found for R74A (11.2 ±1.3), R77A (13.8 ± 1.8), and R82A (2.1 ± 0.8); the b L midpoint was altered in R74A (1022 ± 130), R77A (705 ± 179), and T83A (182 ± 39). The cyt b 561 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 of cyt b 561 , and suggest that structural differences between cyt b 561 and Dcytb in EM5 lead to functional differences between the two cytochromes. Support: NIH grant GM 080575.