Revised Manuscript Received July 10, 1991 abstract: Resonance Raman (RR) spectra are reported for Fe (III), Fe (II), and Fe (II) CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP (MI), cloned in Escherichia coli. The Fe (II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be theHis-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C= C stretching (1620 cm-1) and C^-vinyl bending (402 cm-1). Similar changes are seen for CCP (MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permitsthe His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe (II)-imidazole bond contains components at ca. 235 and 245 cm" 1 for CCP (MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm" 1. Although the Fe (II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP (MI) is photolabile, and the status of the Fe (II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP (MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm" 1 ratio, reflecting a perturbation of the H-bond potential. In the His-52-Leu mutant, a 205-cm" 1 band appears along with the 235/245-cm" 1 doublet at alkalinepH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181-* Gly, Arg-48-* Leu, and Trp-51-*· Phe mutants show an Fe (II)-imidazole stretching mode at 205 cm" 1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond. The conformational changes at alkaline pH apparently exert a stress on the Asp-235-His-175 H-bond that is resisted only if the H-bonding network is intact. The alkaline CO adducts of all mutantsexcept Leu-52 have Fe-CO stretching (RR) and C-0 stretching (IR) frequencies characteristic of normal complexes with neutral imidazole fifth ligands (form F), implying the absence of a strong proximal H-bond. At neutral pH the vibrational frequencies reveal two kinds of CO adduct: linear and ligated by imidazolate (form I), and tilted and H-bonded by a distal group, with a weakened proximal bond (form II). CCP (MI) gives only form I, but the Gly-181 mutant gives a mixture of both forms, reflecting a weakening of the Fe-imidazole bonddue to the loss of an anchoring H-bond between the His-181 imidazole and a propionate substituent of the heme. The Leu-52 mutant also gives a mixture of two forms, but in this case the pH dependence is very weak, and both forms are seen, though with a slightly altered intensity ratio, at alkaline pH. Thus, loss of the His-52 imidazole uncouples the distal H-bonding from the alkaline conformation change. Absorption spectra of the alkaline Fe (III) forms indicate that imidazole is again the sixth ligand, except for the Leu-52 mutant, which binds hydroxide instead, giving a mixed-spin RR spectrum. Lowering the pH produces a 5-c RR spectrum for the Leu-52 mutant, but a mixed spectrum …