Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b561.

Adrenal cytochrome b561 (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb’s function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV–vis absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential ...