Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561

Cytochrome (cyt) b(561) transports electrons across the membrane of chromaffin granules (CG) present in the adrenal medulla, supporting the biosynthesis of norepinephrine in the CG matrix. We have conducted a detailed characterization of cyt b(561) using electron paramagnetic resonance (EPR) and optical spectroscopy on the wild-type and mutant forms of the cytochrome expressed in insect cells. The g(z) = 3.7 (low-potential heme) and g(z) = 3.1 (high-potential heme) signals were found to represent the only two authentic hemes of cyt b(561); models that propose smaller or greater amounts of heme can be ruled out. We identified the axial ligands to hemes in cyt b(561) by mutating four conserved histidines (His54 and His122 at the matrix-side heme center and His88 and His161 at the cytoplasmic-side heme center), thus confirming earlier structural models. Single mutations of any of these histidines produced a constellation of spectroscopic changes that involve not one but both heme centers. We hypothesize that the two hemes and their axial ligands in cyt b(561) are integral parts of a structural unit that we term the "kernel". Histidine to glutamine substitutions in the cytoplasmic-side heme center but not in the matrix-side heme center led to the retention of a small fraction of the low-potential heme with g(z) = 3.7. We provisionally assign the low-potential heme to the matrix side of the membrane; this arrangement suggests that the membrane potential modulates electron transport across the CG membrane.