Magnetic spectroscopic (EPR, ESEEM, Mössbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins

Abstract

Over the past few years extensive studies of model hemes have been carried out that are aimed at understanding the effect of axial ligands and porphyrin substituents on the electronic ground state and detailed magnetic properties of these systems themselves, as well as how they relate to the properties of heme proteins. Using variously substituted iron(III) tetraphenylporphyrins and chlorins, with pyridines, pyrazoles and imidazoles as axial ligands, we have identified three types of low-spin ferriheme centers: type I, which have (d xy ) 2(d xz ,d yz ) 3 electronic ground states with axial ligands aligned in perpendicular planes; type II, which also have (d xy ) 2(d xz ,d yz ) 3 electronic ground states, but with axial ligands aligned in parallel planes; and type III, which have (d xz ,d yz ) 4(d xy ) 1 electronic ground states with axial ligands in any orientation. A subset of the latter type, with axial ligands aligned in parallel planes or with strong macrocycle asymmetry that creates rhombic electron paramagnetic resonance (EPR) spectra, cannot be created using a symmetrical porphyrinate ligand, but rather require a modified or reduced heme ring such as a quinoxalinoporphyrin or chlorin. type I centers are characterized by ‘large g max’ EPR spectra with g>3.3 in most cases and with well-resolved, widely spread magnetic Mössbauer spectra having A zz >800 kG, as well as nuclear magnetic resonance (NMR) spectra that show pyrrole-H resonances in the −10 to −20 ppm range, depending on the temperature, pyrrole-alkyl proton resonances in the +4 to +10 ppm range, meso-H resonances in the +1–4 ppm range, and high intensity NIR magnetic circular dichroism (MCD) spectra. In comparison, type II centers have well-resolved rhombic EPR spectra and less-resolved Mössbauer spectra that are broader on the high-energy side, having A zz in the range 450–560 kG, with a well resolved proton NMR spectra including pyrrole-H resonances in the −15–−30 ppm region, depending on temperature, pyrrole-alkyl proton resonances in the +5 to +16 ppm range, meso-H resonances in the 0–6 ppm range, and medium intensity NIR-MCD spectra. Type III centers have axial EPR spectra with g ⊥=2.6 or smaller and g ‖=0.9–1.95, but with g ‖ often not resolved, and with less-resolved Mössbauer spectra that are broader on the low-energy side and have A zz in the range of 350–450 kG, with well-resolved NMR spectra including pyrrole-H resonances in the 0–10 ppm region, pyrrole-alkyl resonaces in the 4 ppm range, meso-H resonances in the −30–−90 ppm range, depending on temperature, and MCD spectra throughout the UV–vis and NIR range which are extremely weak and difficult to detect. The spectroscopic data from all of these magnetic spectroscopic techniques are self-consistent in defining the orbital of the unpaired electron, and in some cases, the energy separation between the two different electronic ground-states of low-spin ferrihemes. A wide variety of ferriheme proteins also fit this type I–III classification. Electron spin echo envelope modulation (ESEEM) data are useful in defining the orientation of the g-tensor of many of these ferriheme centers. Low-spin iron(III) chlorins are anomalous in exhibiting the spectroscopic properties of a (d xz ,d yz ) 4(d xy ) 1 ground state at 4.2 K (EPR, Mössbauer, MCD), but those of a (d xy ) 2(d xz ,d yz ) 3 ground state at much higher temperatures (−90–+30°C) (NMR).