Relationship between Heme Binding Site Structure and Heme Orientations of Two Ferrocytochrome b₅s. A Study in Prosthetic Group Recognition

The heme binding sites of two b₅ cytochromes, one isolated from beef liver and the other sequentially identical with the globular portion of rat liver cytochrome b₅ and expressed bacterially from a synthetic gene, have been characterized in the diamagnetic reduced state by one-and two-dimensional NMR techniques. Both proteins are heterogeneous in structure in the vicinity of the heme prosthetic group, the result of two possible heme binding orientations. Equilibrium ratios of the two orientations differ between the oxidized beef and bacterially expressed rat proteins, with a ratio of 9:1 of the two isomers in the beef and a 6:4 ratio in the rat. A kinetic (1:1) mixture of the two forms may be trapped by reduction of protein freshly prepared by reconstitution of the apoprotein with hemin. Structural differences between the beef and bacterially expressed rat protein in the vicinity of the heme pocket were determined by nuclear Overhauser effect experiments. Structural differences between the two proteins are discussed in relation to differences in heme orientational equilibria.