Six-coordinated heme groups are involved in a large variety of electron transfer reactions because of their ability to exist in both the ferrous (Fe 2+ ) and ferric (Fe 3+ ) 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 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 ligandsthere 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.

C-type cytochromes with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and over-expressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand that have a considerable impact on both heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by lowtemperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. The Ne c-552 has a ferric low spin (S=1/2) EPR signal characterized by large g anisotropy with g max resonance at 3.34; a similar large g max value EPR signal is found in the mitochondrial Complex III cytochrome c 1 . In Ne c-552, deletion of Asn64 (NeN64Δ) changes the heme ligand-field from more axial to rhombic (small g anisotropy and g max at 3.13) and furthermore hinders the Met fluxionality present in the wild-type enzyme. In Pa c-551 (g max at 3.20) replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g max at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Δ) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Δ, NeG50N/V65Δ, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak axial (Pa c-551) field, but had an impact on Met orientation, fluxionality and relaxation dynamics. Comparison of the electronic fingerprints in the over-expressed proteins and their mutants reveals a linear relation between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III) the large g max value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.

C-type cytochromes with histidine-methionine (His-Met) iron coordination play important roles in electron-transfer reactions and in enzymes. Low-temperature electron paramagnetic resonance (EPR) spectra of low-spin ferric cytochromes c can be divided into two groups, depending on the spread of g values: the normal rhombic ones with small g anisotropy and g(max) below 3.2, and those featuring large g anisotropy with g(max) between 3.3 and 3.8, also denoted as highly axial low spin (HALS) species. Herein we present the detailed magnetic properties of cytochrome c(553) from Bacillus pasteurii (g(max) 3.36) and cytochrome c(552) from Nitrosomonas europaea (g(max) 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c(553) shows the presence of a minor rhombic species at pH 6.2 (6 %), whereas cytochrome c(552) has about 25 % rhombic species over pH 7.5. The detailed Mössbauer analysis of cytochrome c(552) confirms the presence of these two low-spin ferric species (HALS and rhombic) together with an 8 % ferrous form with parameters comparable to the horse cytochrome c. Both EPR and Mössbauer data of axial cytochromes c with His-Met iron coordination are consistent with an electronic (d(xy))(2) (d(xz))(2) (d(yz))(1) ground state, which is typical for Type I model hemes.

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