HEME-PROTEIN INTERACTIONS IN CYTOCHROME-C PEROXIDASE REVEALED BY SITE-DIRECTED MUTAGENESIS AND RESONANCE RAMAN-SPECTRA OF ISOTOPICALLY LABELED HEMES

Isotope labeling has been used to assign the resonance Raman spectra o f cytochrome c peroxidase, expressed in Escherichia coli [CCP(MKT)], a nd of the D235N site mutant. Fe-54 labeling establishes the coexistenc e of two separate bands (233 and 246 cm(-1)), arising from the stretch ing of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His 175 imidazole NdeltaH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other t he proton is transferred to the carboxylate. When Asp235 is replaced b y Asn, the H-bond is lost, and the Fe-His stretching frequency is mark edly lowered. Two new RR bands are produced, at 205 and 185 cm(-1), as a result of coupling between the shifted Fe-His vibration and a nearb y porphyrin mode; the two bands share the Fe-54 sensitivity expected f or Fe-His stretching. C=C stretching and CbetaC = C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the pro toheme prosthetic group via selective vinyl deuteration. In the acid f orm of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm(-1) for the C=C stretch, and at 406 cm(-1) for the CbetaC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline f orm of the enzyme, to 1628 cm(-1) for C=C stretching, and to 418 cm(-1 ) for CbetaC = C bending, while the C-vinyl frequencies remain unshift ed. Thus, the acid alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This pertu rbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. T he perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CbetaC=C bending frequency is shifted to a smaller extent (412 cm(-1)). This at tenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound. (C) 1996 Jo hn Wiley & Sons, Inc.