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The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and non-native states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed IIIh, which is structurally different from the alkaline states, TV1 and TV2, contrary to current belief. At neutral or slightly acidic pH, IIIh is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm-1, which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, IIIh, does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe3+-M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice. [ABSTRACT FROM AUTHOR]

The reaction between cytochrome f and plastocyanin is a central feature of the photosynthetic electron-transport system of all oxygenic organisms. We have studied the reaction in solution to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. In a previous publication [Schlarb-Ridley, B. G., et al. (2003) Biochemistry 42,4057-4063], we suggested that the reaction is diffusion-controlled because of a strong effect of viscosity of the medium. The effects of viscosity and temperature have now been examined in detail. High molecular mass viscogens (Ficoll 70 and Dextran 70), which might mimic in vivo conditions, had little effect up to a relative viscosity of 4. Low molecular mass viscogens (ethane diol, glycerol, and sucrose) strongly decreased the bimolecular rate constant (k2) over a similar viscosity range. The effects correlated well with the viscosities of the solutions of the three reagents but not with their dielectric constants or molalities. A power law dependence of k2 on viscosity suggested that k2 depends on two viscosity-sensitive reactions in series, while the reverse reactions are little affected by viscosity. The results were incompatible with diffusion control of the overall reaction. Determination of the effect of temperature on k2 gave an activation enthalpy, Δwhich is also incompatible with diffusion control. The results were interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer. [ABSTRACT FROM AUTHOR]