An extensive series of five-and six-coordinate (1-methylimidazo1e)-, (1-butylamine)-, and (pyridine)iron(II, 111) chlorin complexes are characterized for the first time with magnetic circular dichroism (MCD) spectroscopy. The chlorins (dihydroporphyrins) employed are octaethylchlorin, mesochlorin, and "methyl"ch1orin (2,2,4-trimethyldeuterochlorin, featuring a gem-dimethyl-substituted peripheral carbon). The species studied include the bis( 1-methylimidazole)ferric, bis( 1 -methylimidazole)ferrous, (1,2-dimethylimidazole)ferrous, and 1-methylimidazole/ CO or NO complexes together with analogous bis(pyridine)-and bis( 1-buty1amine)ferrous adducts and their COligated derivatives. The results presented establish MCD spectral signatures for use in determining whether an iron chlorin-containing protein bears either a histidine or a lysine axial ligand. Analysis of data obtained with different ring-reduced mesochlorin isomers shows that the method is insensitive to the site of pyrrole ring reduction. This latter observation will facilitate the use of mesochlorins in reconstitution experiments with structurally-defined heme proteins in order to investigate mixed-ligand complexes which are difficult to generate synthetically. In general, the MCD spectra of iron chlorin complexes are most sensitive to the identity, number, and type of axial ligand, along with the oxidation and spin state, and are relatively insensitive to changes in the equatorial plane such as the site of pyrrole ring reduction. Therefore, it appears that MCD spectroscopy will be of particular use in the identification of proximal and distal axial ligands in chlorin-containing proteins, as has repeatedly been shown to be the case with protoheme-based iron porphyrins.
To probe the identity of the active site hemetype prooshetic group ofmyeloperoxidase, whose (5,6,9,10), the spectroscopic properties of analogous derivatives with the same oxidation and ligation states can be used to probe the similarity or difference in their prosthetic group structures. MCD spectral comparison between myoglobin reconstituted with Spirographis heme (Spirographis Mb) and MPO in this study reveals considerable spectral similarities between parallel derivatives of these two proteins, especially in the ferric high-spin native form and the ferrous low-spin complexes. Given the repeatedly demonstrated ability of MCD spectroscopy to identify or distinguish two apparently similar chromophores more definitively than electronic absorption spectroscopy (18-20), the present finding provides strong support for a formyl-substituted porphyrin as the structure of the prosthetic group macrocycle of MPO. EXPERIMENTAL PROCEDURES Spirographis Mb was prepared from horse heart apo-Mb and Spirographis heme as described by Sono and Asakura (16,21,22). MPO was purified from bovine spleen as described (23). Metal-free trans-octaethylchlorin (GEC) was obtained by a published method (24) and metallated to produce Fe-(OEC)Cl using a modification (25) of the direct iron insertion method (26). All chemicals were reagent grade and were obtained from Aldrich or Sigma. N-Methylimidazole and benzene were further purified by distillation.The reduced pyridine hemochromogen [the ferrous bis(pyridine) adduct] of Spirographis heme (and Mb) or MPO was generated by dissolving the heme or diluting the concentrated protein stock solution into a pyridine/NaOH solution [25-35% pyridine (vol/vol) and 0
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.