RESONANCE RAMAN-SPECTRA AND THE ACTIVE-SITE STRUCTURE OF SEMISYNTHETIC MET80CYS HORSE HEART CYTOCHROME-C

Resonance Raman (RR) spectra are reported for semisynthetic Met80Cys horse heart cytochrome c. For the Fe(III) form, the porphyrin high-frequency skeletal modes are at positions close to those of the imidazole adduct of cytochrome P450, confirming earlier spectroscopic indications that the Cys80 side chain is bound to the Fe3+ as a thiolate anion. The low-frequency RR spectrum shows changes indicating a relaxation of the protein-induced heme distortion present in native cyt c; a change in the protein fold is suggested, due to the shortening of the ligating side chain from methionine to cysteine. When the (FeCO)-C-II adduct of the cyt c-Cys80 mutant is photolyzed, the product contains low-spin heme, implying religation by an endogenous ligand. The position of the nu(11) porphyrin back-bending marker indicates that the endogenous ligand donor strength is greater than that of methionine but less than that of lysine. Cys80 ligation as a neutral thiol is suggested, with proton uptake accompanying the Fe(III)/Fe(II) reduction. Two forms of the (FeCO)-C-II adduct, A(0) and A(-1), were detected, with RR nu(FeC) bands at 486 and 491 cm(-1) and IR nu(CO) bands at 1966 and 1981 cm(-1). These pairs of frequencies are located in the nu(FeC)/nu(CO) back-bonding graph near the line characteristic of imidazole (rather than thiolate) ligation. This behavior is consistent with CO displacement of the Cys80 ligand, but His18 substitution cannot be ruled out if Cys80 is bound as thiol, since its donor strength might then be comparable to that of imidazole. The frequencies of form A(0), nu(FeC) = 486 cm(-1) and nu(CO) = 1966 cm(-1), are characteristic of CO bound in a hydrophobic pocket, while those of form A(-1), nu(FeC) = 491 cm(-1) and nu(CO) = 1981 cm(-1), indicate significant inhibition of back-bonding, perhaps via an interaction with a lone pair of the displaced endogenous ligand, either Cys80 or His18.