CONFORMATIONAL CHANGE AND HISTIDINE CONTROL OF HEME CHEMISTRY IN CYTOCHROME-C PEROXIDASE - RESONANCE RAMAN EVIDENCE FROM LEU-52 AND GLY-181 MUTANTS OF CYTOCHROME-C PEROXIDASE

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 the His-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-beta-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 permits the 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 alkaline pH, 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 mutants except Leu-52 have Fe-CO stretching (RR) and C-O stretching (IR) frequencies characteristic of normal complexes with neutral imidazole fifth ligands (form I'), 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 bond due 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 for the Gly-181 mutant, with contributions from high- and low-spin 6-c as well as 5-c heme. This effect is again attributed to the loss of the anchoring His-181-propionate interaction, which allows the Fe(III) to more readily bind a water molecule. At low pH values, the Leu-52 mutant shows some 6-c high-spin contribution, as does CCP(MI) itself, but this contribution is buffer-dependent, being somewhat greater for acetate than for citrate. The only mutant failing to show a low pH 6-c contribution is Leu-48, suggesting that the Arg-48 side chain plays an obligatory role in the binding of a sixth ligand, either water or a buffer anion. Since the appearance of a 6-c low pH component has been linked to aging of the protein in the case of baker's yeast CCP, it may be that an alteration which increases the Arg-48 mobility is involved. Functional implications of the distal and proximal interactions and of the acid/alkaline conformation change are discussed.