THE ORIGIN OF DIFFERENCES IN THE PHYSICAL-PROPERTIES OF THE EQUILIBRIUM FORMS OF CYTOCHROME B(5) REVEALED THROUGH HIGH-RESOLUTION NMR STRUCTURES AND BACKBONE DYNAMIC ANALYSES

On the basis of a comparison of high-resolution solution structures ca lculated for both equilibrium forms of rat ferrocytochrome bs, differe nces in reduction potential and thermodyanmic stability have been char acterized in terms of significant structural and dynamic differences b etween the two forms. The dominant difference between A and B conforma tions has long been known to be due to a 180 degrees rotation of the h eme in the binding pocket about an axis defined by the alpha- and gamm a-meso carbons, however, the B form has not been structurally characte rized until now. The most significant differences observed between the two forms were the presence of a hydrogen bond between the 7-propiona te and the S64 amide in the A form but not the B form and surprisingly a displacement of the heme out of the binding pocket by 0.9 iq in the B form relative to the A form. The magnitude of other factors which c ould contribute to the known difference in reduction potentials in the bovine protein [Walker, F. A., Emrick, D., Rivera, J. E., Hanquet, B. J., and Buttlaire, D. H. (1988) J. Am. Chern. Sec. 110, 6234-6240], s uch as differences in the orientation of the axial imidazoles and diff erences in hydrogen bond strength to the imidazoles, have been evaluat ed. The dominant effector of the reduction potential would appear to b e the lack of the hydrogen bond to the S64 amide in the B form which f rees up the propionate to charge stabilize the iron in the oxidized st ate and thus lower the reduction potential of the B form. The structur e we report for the A form, based on heteronuclear NMR restraints, inv olving a total of 1288 restraints strongly resembles both the X-ray cr ystal structure of the bovine protein and a recently reported structur e for the A form of the rat protein based on homonuclear data alone [B anci, L., Bertini, I., Ferroni, F., and Rosato, A. (1997) fur. J. Bioc hem. 249, 270-279]. The rmsd for the backbone atoms of the A form is 0 .54 Angstrom (0.92 Angstrom for all non-hydrogens). The rmsd for the b ackbone of the B form is 0.51 Angstrom (0.90 Angstrom for all non-hydr ogen atoms). An analysis of backbone dynamics based on a model-free an alysis of N-15 relaxation data, which incorporated axially symmetric d iffusion tensor modeling of the cytochrome, indicates that the protein is more rigid in the reduced state relative to the oxidized state, ba sed on a comparison with order parameters reported for the bovine prot ein in the oxidized state [Kelly, G. P., Muskett, F. W., and Whitford, D. (1997) fur. J. Biochem. 245, 349-354].