Modulation of the Ligand-Field Anisotropy in a Series of Ferric Low-Spin Cytochrome c Mutants derived from Pseudomonas aeruginosa Cytochrome c-551 and Nitrosomonas europaea Cytochrome c-552 : A Nuclear Magnetic Resonance and Electron Paramagnetic Resonance Study

Cytochromes of the c type 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 overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with gmax resonance at 3.34; a similar large gmax value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Δ) changes the heme ligand field from more axial to rhombic (small g ...