Solution structure of oxidized microsomal rabbit cytochrome b(5) - Factorsdetermining the heterogeneous binding of the heme

Cytochrome b(5) is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the alpha and gamma meso protons. For rabbit cytochrome b(5 ), the A/B ratio is 5: 1. The solution structure of the major form of the o xidized soluble fragment of rabbit microsomal cytochrome b(5) (94 amino aci ds) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 (3)J couplings, a family of 40 energy-minimized conformers were obtained with average back bone rmsd (for residues 4-84) of 0.060 +/- 0.016 nm and average target func tion of 0.0078 nm(2), no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5: 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into th e factors determining the distribution in solution of the two isomers diffe ring with respect to heme orientation. It appears that residues 23 and 74 a re both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interac tions are the key factors in determining the relative stability of one isom er with respect to the other.