EFFECT OF AXIAL LIGAND PLANE REORIENTATION ON ELECTRONIC AND ELECTROCHEMICAL PROPERTIES OBSERVED IN THE A67V MUTANT OF RAT CYTOCHROME B(5)

Mutational studies directed at evaluating the effect of the axial liga nd plane orientation on electrochemical properties of cytochrome b(5) have been performed. As described in the previous paper, structural co nsequences of one of these mutations, the A67V mutation, have been eva luated using NMR solution methods. The lack of large shifts relative t o the wild-type protein in both the imidazole N delta nitrogen and pro ton resonances of the H63 imidazole ring indicates that the hydrogen b ond between the carbonyl of F58 and the imidazole ring of H63 remains intact in this mutant. Effects of the imidazole plane reorientation on the Fe d-orbitals were evaluated on the basis of interpretation of EP R spectra, near-infrared bands associated with ligand-to-metal charge transfer transitions, reorientation of the anisotropy of the paramagne tic center determined by calculation of pseudocontact shifts, and the temperature dependence of the contact-shifted resonances. The dominant effect of the imidazole reorientation appears to have been a destabil ization of the d(xz) orbital energy and a reorientation of the d(pi) o rbitals. This is surprising in light of the -20 mV shift in the reduct ion potential of the mutant relative to the wild-type protein and indi cates that a destabilization of d(yz)-orbital energy level of the redu ced state dictates the observed change in reduction potential. Measure d values for the reorganizational energy and heterogeneous electron tr ansfer rates were indistinguishable for wild-type and mutant proteins. This is perhaps surprising, given significant differences in the patt ern of electron delocalization into the porphyrin ring observed as sig nificantly altered contact shift patterns. Mutational studies perturbi ng the H39 imidazole were also performed but with more limited success .