Six‐coordinated heme groups are involved in a large variety of electron transfer reactions because of their ability to exist in both the ferrous (Fe²⁺) and ferric (Fe³⁺) state without any large differences in structure. Our studies on hemes coordinated by two histidines (bis‐His) and hemes coordinated by histidine and methionine (His‐Met) will be reviewed. In both of these coordination environments, the heme core can exhibit ferric low spin (electron paramagnetic resonance EPR) signals with large g_max values (also called Type I, highly anisotropic low spin, or highly axial low spin, HALS species) as well as rhombic EPR (Type II) signals. In bis‐His coordinated hemes rhombic and HALS envelopes are related to the orientation of the His groups with respect to each other such that (i) parallel His planes results in a rhombic signal and (ii) perpendicular His planes results in a HALS signal. Correlation between the structure of the heme and its ligands for heme with His‐Met axial ligation and ligand‐field parameters, as derived from a large series of cytochrome c variants, show, however, that for such a combination of axial ligands there is no clear‐cut difference between the large g_max and the “small g‐anisotropy” cases as a result of the relative Met‐His arrangements. Nonetheless, a new linear correlation links the average shift 〈δ〉 of the heme methyl groups with the g_max values. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1064–1082, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

High resolution crystal structures of myoglobin in the pH range 5.2-8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe-O bond length (1.86-1.90 A) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe-O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an Fe(IV)-O species with a single bond. The presence of Fe(IV) is indicated by a small isomer shift of delta = 0.07 mm/s from Mössbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.