It has been well established that the heme redox potential is affected by many different factors. Among others, it is sensitive to the proximal heme ligand and the conformation of the propionate and vinyl groups. In the cytochrome P450 BM3 heme domain, substitution of the highly conserved phenylalanine 393 results in a dramatic change in the heme redox potential [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429]. We have used resonance Raman spectroscopy to characterize heme structural changes and modification of heme interactions with the protein matrix that are induced by the F393 substitutions and to determine their correlation with the heme redox potential. Our results show that the Fe-S stretching frequency of the 5-coordinated, high-spin ferric heme is not affected by the mutations, suggesting that the electron density in the Fe-S bond in this state is not affected by the F393 mutation and is not a good indicator of the heme redox potential. Substrate binding perturbs the hydrogen bonding between one propionate group and the protein matrix and correlates to both the size of residue 393 and the heme redox potential. However, heme reduction does not affect the conformation of the propionate groups. Although the conformation of the vinyl groups is not affected much by substrate binding, their conformation changes from mainly out-of-plane to predominantly in-plane upon heme reduction. The extent of these conformational changes correlates strongly with the size of the 393 residue and the heme redox potential, suggesting that steric interaction between this residue and the vinyl groups may be of importance in regulating the heme redox potential in the P450 BM3 heme domain. Further implications of our findings for the change in redox potential upon mutation of F393 will be discussed.

Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.