Low-temperature EPR and Mössbauer spectroscopy of two cytochromes with His-Met axial coordination exhibiting HALS signals

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 c₅₅₃ from Bacillus pasteurii (g_max 3.36) and cytochrome c₅₅₂ from Nitrosomonas europaea (g_max 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c₅₅₃ 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 c₅₅₂ 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.