Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
Cytochrome b5 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 b5, the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3J couplings, a family of 40 energy-minimized conformers were obtained with average backbone rmsd (for residues 4-84) of 0.060 +/- 0.016 nm and average target function of 0.0078 nm2, 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 the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other.
Banci
L
L
Department of Chemistry and Centro di Risonanze Magnetiche, University of Florence, Sesto Fiorentino, Italy.
Bertini
I
I
Rosato
A
A
Scacchieri
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000737:chemistry; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D013045:Species Specificity
D011166:Porphyrins; D011994:Recombinant Proteins; D012996:Solutions; D006418:Heme; D015786:Cytochromes b5