Protonation of the Cysteine Axial Ligand Investigated in His/Cys c-type Cytochrome by UV-Vis, Mid- and Far-IR Spectroscopy.

His/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Towards better understanding Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and Mid to Far FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C = -375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys ν(SH), His ν(C5N1), heme δ(CmH)) and Far-IR (ν(Fe(II)-His), ν(His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination.