The M80A variant of yeast i so- I -cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and 02 concentration. The E degrees' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately-200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11 The invariance of the electrochemical features up to pH I I indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O-2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O-2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.