Urea-induced denaturation of immobilized yeast iso-1 cytochrome c: Role of Met80 and Tyr67 in the thermodynamics of unfolding and promotion of pseudoperoxidase and nitrite reductase activities

Electron Transfer and Electrocatalytic Properties of the Immobilized Met80Ala Cytochrome c Variant in Dimethylsulfoxide

“…[1,[38][39][40][41] This variant is nontrimethylated and carries the C102T mutation to prevent protein dimerization. [1,[40][41][42] Protein solutions were made up in 5 mM Tris-HCl and NaCl at pH 7.5 for storage at À 20°C. All chemicals were reagent grade.…”

“…A coverage of 17.7 � 0.8 pmol cm À 2 was determined, in line with previous results. [42] Cyclic voltammograms at variable scan rate were recorded to determine the rate constant k s for the interfacial electrochemical ET process for the adsorbed protein, according to Laviron. [48] Effects of uncompensated cell resistance were minimized using the positivefeedback iR compensation function of the potentiostat, set at a value slightly below that at which current oscillations emerge.…”

“…[59] For an immobilized protein, however, an increase in folding stability can occur, as observed for M80A immobilized on anionic SAM towards urea denaturation. [42] At high DMSO concentrations, the observed major structural effects could include a change in axial heme iron ligation, such as the replacement of the (6th) hydroxide ion (that binds to the heme iron of M80 A in water) with a lysine or a histidine residue. Analogous transitions involving the 6th axial ligand switch were observed previously for freely diffusing M80 A in the presence of a denaturing agent (urea), [60] for the hydrophobically electrode-immobilized protein or in the presence of cardiolipin liposomes.…”

“…[32,42,61,73,74] The latter term in fact is correlated to the effects of the surface on the reorganization energy, the tunneling factor β and the electron transfer heme-electrode distance. [42,61,[68][69][70][75][76][77] In the absence of DMSO, M80A shows a significantly lower k s value than that for wt ycc [1,41] due to the increase in ΔH # and, to a larger extent, the decrease in A (Table 2). This effect appears to be mainly related to the specific nature of the M80 A mutation because for His/Met-ligated species bearing mutations on surface charged residues, the contributions of ΔH # and A to the decrease in k s are comparable.…”

“…The changes in the contributions of ΔH # and A to k s induced by DMSO below 60 % (Figure 3B, C) parallel those for immobilized M80 A in the presence of increasing concentrations of unfolding agent (urea). [42] The increase in A suggests that the presence of DMSO alters the structural constraints of M80 A that control the heterogeneous electron transfer, possibly modifying the composition of the solvent near the metal center, the H-bonding network and the size of the heme crevice. The same factors would induce the increase in ΔH # , which is responsible for the decrease in k s .…”