Iron Chlorin-Reconstituted Histidine-Ligated Heme Proteins as Models for Naturally Occurring Iron Chlorin Proteins: Magnetic Circular Dichroism Spectroscopy as a Probe of Iron Chlorin Coordination Structure

Apomyoglobin, horseradish peroxidase, and cytochrome b5 have been successfully reconstituted with several iron chlorin prosthetic groups. The resulting green iron chlorin-bound derivatives have a histidine proximal ligand and therefore serve as models for naturally occurring histidine-ligated iron chlorin proteins. Characterization of these systems with electronic absorption and magnetic circular dichroism (MCD) spectroscopy has established definitive and diagnostic spectral signatures for both ferric and ferrous iron chlorin states. As with regular iron heme systems, MCD spectroscopy is sensitive to changes in the coordination structure, spin state and oxidation state of iron chlorin systems, while the nature of the protein environment or solvent does not contribute significantly to the band pattern. The MCD signals of the iron chlorin-reconstituted proteins were generally found to be broad and reduced in intensity and to have different line shapes when compared to the intense and often symmetric bands seen for iron porphyrin systems. This reflects the reduction in symmetry of the chlorin macrocycle. The MCD spectra of the iron chlorin systems are also distinct from those previously reported for the other type of green iron heme, namely iron formyl-substituted porphyrins. A significant change in the band pattern of the MCD spectra is associated with changes from high-spin to low-spin in the ferric iron chlorin complexes examined and from ferric to ferrous oxidation states in low-spin complexes such as iron chlorin-reconstituted cytochrome b5, a bis(imidazole)-ligated species. The spectra of low-spin ferrous chlorin complexes with π-acceptor ligands such as CO or NO trans to histidine are also distinguishable from those of complexes with imidazole, a σ-donor, in the trans position. These results provide spectroscopic models for naturally-occurring iron chlorin proteins bearing a proximal histidine ligand and demonstrate the utility of MCD spectroscopy in the determination of iron chlorin coordination structure. In addition, MCD spectroscopy can readily distinguish the two green heme systems, iron chlorins and iron formyl-substituted porphyrins. © 1994, American Chemical Society. All rights reserved