Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561

Cytochromes b561 (Cyts b561) are ubiquitous membrane proteins catalyzing ascorbate-mediated trans-membrane electron transfer. A heterologous expression system in Saccharomyces cerevisiae was developed to study their structure–function relationship. Recombinant mouse chromaffin granule Cyt b561 (CGCytb) shows spectral characteristics, ascorbate reducibility, and redox potentials identical to that of the native bovine protein. Moreover, the reconstituted recombinant protein mediated trans-membrane electron transport with kinetic characteristics similar to that of bovine CGCytb. Site-directed mutant analysis supports the presence of two hemes coordinated by the highly conserved His pairs H52/H120 and H86/H159. Reduction of CGCytb by ascorbate showed biphasic kinetics (Kd1: 0.016 ± 0.005 mM, Kd2: 1.24 ± 0.19 mM). Mutation of a well-conserved Arg residue (R72) abolished high affinity CGCytb reduction by ascorbate, indicating that this residue may be critical for substrate binding. On the other hand, mutation of a Lys previously suggested to play a role in ascorbate binding (K83), did not affect the ascorbate-mediated reduction of the protein.