Redox Thermodynamics of the Native and Alkaline Forms of Eukaryotic and Bacterial Class I Cytochromes c

Abstract

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

Redox thermodynamics of cytochromes c subjected to urea induced unfolding

“…/MV ? couple (MV = methylviologen) [33]. All the redox potentials reported here are referred to SHE.…”

“…0.01 M NaCl ? 0.01 M phosphate buffer at pH 7 were used as base electrolytes and urea concentrations were varied between 0 and 10 M. The formal reduction potentials E°0 were calculated from the average of the anodic and cathodic peak potentials [33] and found almost independent of scan rate in the range 0.02-2 V s -1 . In all cases the linear dependence of i pc on v 1/2 (v = scan rate) indicates a diffusion controlled electrochemical process.…”

“…In all cases the linear dependence of i pc on v 1/2 (v = scan rate) indicates a diffusion controlled electrochemical process. For each species, the experiments were performed at least five times and the reduction potentials were found to be reproducible within ±0.002 V. The CV experiments at different temperatures were carried out with a cell in a ''nonisothermal'' setting [33], namely in which the reference electrode was kept at constant temperature (21 ± 0.1°C) whereas the half-cell containing the working electrode and the Vycor Ò junction to the reference electrode was under thermostatic control with a water bath. The temperature was varied from 5 to 55°C.…”

“…The temperature was varied from 5 to 55°C. With this experimental configuration, the reaction entropy for reduction of the oxidized protein (DS 0 rc ) is given by [33][34][35]:…”

“…Under the same assumption, the enthalpy change (DH 0 rc ) was obtained from the Gibbs-Helmholtz equation, namely as the negative slope of the E°0/T versus 1/T plot. The nonisothermal behavior of the cell was checked carefully by determining the DH 0 rc and DS 0 rc values of the ferricyanide/ferrocyanide couple [33][34][35].…”