Redox properties of wild-type and heme-binding loop mutants of bacterial cytochromes C measured by direct electrochemistry.

We have used protein film voltammetry (PFV) to determine the midpoint potentials of the Pseudomonas aeruginosa, Hydrogenobacter thermophilus, and Nitrosomonas europaea wild-type monoheme cytochromes c (cyts c; PA, HT, and NE, respectively), as well as PA N64Q, HT Q64N, and NE V65delta mutants, as a function of pH, and buffer conditions. Recent studies have suggested that the identity of the 64 position of the heme-binding loop (either Asn or Gln) strongly influences the conformation of the Met ligand that binds the heme iron. The PFV studies reveal that HT and NE possess significantly lower potentials (wild-type cyts c having E(m) values of +227 and +250 mV vs SHE) than PA (+290 mV) in 50 mM phosphate buffer, pH 7 at 3 degrees C. The HT Q64N mutant rises in potential compared to wild-type, and the PA N64Q mutant has a lower potential, indicating relationships between Met ligand fluxion, hydrogen bonding to the Met ligand, and redox chemistry. Surprisingly, NE V65delta, possessing a heme binding loop nearly identical to that of the PA protein, displayed an E(m) of +232 mV, even lower than wild-type NE. These data are discussed in terms of models of Met ligand properties and proton dependence.