Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

The M80A variant of yeast iso-1-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 O2 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 11 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 O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.

Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinating Ala residue and alteration of the hydrogen bonding network in the region nearby following substitution of Tyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electron transfer (ET) and the heme-mediated electrocatalytic reduction of H(2)O(2). To this end, the voltammetry of the Met80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbed on carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxide concentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67 results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects on the E degrees' values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V (vs SHE). On the contrary, both double variants show much lower ET rates compared to Met80Ala, most likely as a consequence of a change in the ET pathways. In the present nondenaturing immobilizing conditions, and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H(2)O(2) reduction at the electrode, whereas wild-type cytochrome c does not. H(2)O(2) electrocatalysis occurs with an efficient mechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resulting dioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presence of a general acid-base residue for H(2)O(2) recognition and binding through H-bonding in the distal heme site is a key requisite for the reductive turnover of this substrate.

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for the corresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM. For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.