A systematic investigation was carried out concerning relationships between visible and Soret spectra of heme complexes and the nature of the axial ligands. Dioxygen (O2), carbon monoxide (CO), and five-coordinate complexes were prepared from proto-, meso-, and 2,4-dimethyldeuteroheme dimethyl esters in NN-dimethylformamide (DMF) solution. A variety of axial ligands was employed, including imidazoles, pyridines, aliphatic amines, and very weak bases such as DMF and acetone. Variations in the ligands included differences in basicity and differences in substituents which sterically hindered coordination to heme iron. In the five-coordinate complexes, a shift to shorter wavelength in both the visible and Soret peaks accompanied a change to a more hindered axial ligand. Difference spectra obtained by taking the spectrum of a heme complex with a more hindered ligand minus that of a less hindered ligand approximated the T minus R state difference spectrum reported by Perutz et al, [Perutz, M. F., Ladner, J. E., Simon, S. R., & Ho, C. (1974) Biochemistry 13, 2163] for deoxyhemoglobin (deoxy-Hb) Kempsey ± inositol hexaphosphate (IHP). In the CO complexes, a decrease in the basicity of the ligand or an increase in the steric hindrance of the axial ligand also resulted in a blue-shifted λmax accompanied by an increase in the ratio of intensities of the long-wavelength visible peak (a) to that of the shorter wavelength visible peak (β). The latter parameter is termed the α/β ratio. Spectra of the O2 complexes were observed only at temperatures below -40 °C because of limited stability at higher temperatures. In O2 complexes, shifts in λmax and changes in the α/β ratio with unhindered ligands showed much the same pattern as with the CO complexes, but hindered ligands such as 1,2-dimethylimidazole and 1,2,4,5-tetra-methylimidazole gave red-shifted visible λraax and low α/β ratios compared to the unhindered O2-heme-l-n-butyl-imidazole complex. This observation is interpreted as being due to the greater ease with which the Fe is distorted from the porphyrin plane in O2 complexes than in CO complexes. Comparison of α/β ratios of model O2- and CO-protoheme complexes with those of hemoglobins and myoglobins formed the basis for the suggestion that the proximal histidine is restrained by the protein, producing a relatively weak axial Fe-N interaction, both in liganded R-state hemoglobins and in common myoglobins. T minus R state difference spectra reported by Perutz et al. [Perutz, M. F., Kilmartin, J. V., Nagai, K., Szabo, A., & Simon, S. R. (1976) Biochemistry 15, 378] for O2- and CO-Hb Kansas ± IHP were approximated by model protoheme complexes of a stronger minus a weaker axial ligand. These results are interpreted in terms of T-state steric conflict between coordinated O2 or CO and amino acid side chains on the distal side of the heme. Transition to T state would result in the heme complex being forced toward the proximal imidazole, thereby strengthening the axial Fe-N bonding interaction and introducing strain in both the heme complex and the protein. © 1979, American Chemical Society. All rights reserved.
