The Function of Tyrosine 74 of Cytochrome b5

Tyrosine 74, which is part of a hydrophobic patch on the surface of rat cytochrome b5, also forms van der Waals contacts with the heme prosthetic group of the protein. In addition it is a member of an aromatic network of amino acids which includes Phe-35 and the axial ligand, His-39. Because of its strategic location in the protein, the Tyr-74 residue was mutated to a lysine in order to investigate how it affected the interaction of heme with the protein and whether it might be an alternative binding site and an electron transfer path which cytochrome b5 would use with its amphipathic electron transfer partners cytochrome P450 and its corresponding NADPH cytochrome P450 reductase. The mutant protein receives electrons from NADPH cytochrome P450 reductase and provides the second electron to cytochrome P450 to catalyze the metabolism of methoxyflurane, a substrate which requires cytochrome b5 for its metabolism, at the same rate as the wild type protein. The Tyr74Lys mutant exhibits a normal redox potential and spectroscopic properties identical to those of the wild type protein. Under equilibrium conditions in the presence of urea, heme dissociation and denaturation occur simultaneously with a free energy of 3.4 kcal/mol. The free energy of activation of heme dissociation from the wild type protein is 22.7 kcal/mol. The free energy and free energy of activation of the Tyr74Lys mutant are 1.4 kcal/mol less than the wild type values, indicating that the mutant binds heme 10-fold less tightly and dissociates heme 10 times faster than the wild type protein. Heme transfer experiments demonstrate that heme spontaneously dissociates 6 times faster from the mutant than the wild type protein (t1/2 1.9 and 11.5 h, respectively). The most likely conformation of Lys-74 in the mutant protein was determined by calculating the Lys-74 rotamer with the minimal energy using an energy-based conformation search method. This conformation was subsequently modeled on the computer graphics. Not unexpectedly, the side chain of Lys-74 is shifted toward the surface of the protein to allow solvation of the positive charge on the ϵ amino group of lysine. This movement of the lysine residue results in the formation of a cavity on the surface of the cytochrome b5 molecule, which exposes a heme methyl and vinyl group to aqueous solvent thereby destabilizing the binding between the protein and its hydrophobic prosthetic heme group.