Oxidation state-dependent folding and stability of cytochrome c

Oxidation state-dependent reversible folding and unfolding of cytochrome c has been studied by equilibrium and kinetic methods using NMR and optical probes. At equilibrium, unfolding of the oxidized protein is less cooperative than the reduced protein. Cytochrome c is more stable in the reduced state than in the oxidized state by approximately 7 kcal mol$\sp{-1}$. The difference in Gibbs free-energy between the two redox states originate from a cumulative effect of contributions from the Fe-M80 bond energy, the heme solvation energy, and electrostatic, conformational, and van der Waals interactions. Kinetically, reduced cytochrome c refolds fast ($\tau\approx8\pm3$ ms) in an apparent two-state process. Refolding of oxidized cytochrome c, near neutral pH, proceeds via an intermediate ($\tau\approx20$ ms) carrying a configurationally frustrated chain segment. To resolve the redox-related properties and the kinetic differences of folding, intrapolypeptide interactions were studied in denatured/unfolded states. Upon unfolding M80 is invariably de-ligated from the sixth coordination site of the iron. Intramolecular ligands, H26 and H33 in the oxidized state, and H26, H33, M65, and M80 in the reduced state, make contact with the heme at this axial site. The equilibrium fractions of five-coordinated heme are $0.6\times10\sp{-5}$ and 0.45 for the oxidized, and the reduced, respectively. The rates of ligand exchange with the reduced heme site are faster on the folding time scale. Thus, refolding of reduced cytochrome c is not affected by the type of ligands and their dissociation rates. In the oxidized state, the dissociation rates of the two non-native histidines are slow on the folding time scale. During folding the histidines become trapped in the protein interior, pulling the H26- and H33-resident portion of the polypeptide away from the distal side of the heme to the proximity of M80. This geometric frustration slows down folding of oxidized cytochrome c at later stages. Under mildly denaturing conditions the M80 ligand of reduced cytochrome c is replaceable with CO. The N$\Leftrightarrow$U equilibrium is thus tipped to the right by $\approx$5.9 kcal mol$\sp{-1}$ which is equivalent to the CO binding energy. Photodissociation of the CO then causes refolding. This experiment will be most useful in studying intramolecular chain dynamics in the unfolded states