Folding of horse cytochrome c in the reduced state

Equilibrium and kinetic folding studies of horse cytochrome c in the reduce d state have been carried out under strictly anaerobic conditions at neutra l pH, 10 degreesC, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Sore t heme absorbance, excitation energy transfer from the lone tryptophan resi due to the ferrous heme, and far-UV circular dichroism (CD) are all biphasi c and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these trans itions yielded DeltaG(H2O) = 18.8(+/-1.45) kcal mol(-1), and C-m = 5.1(+/-0 .15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cy tochrome c and the known stability of oxidized cytochrome c to estimate a v alue of -164 mV as the redox potential of the unfolded protein. Stopped-flo w kinetics of folding and unfolding have been recorded by Soret heme absorb ance, and tryptophan fluorescence as observables. Th refolding kinetics are monophasic in the transition region, but become biphasic as moderate to st rongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U <----> N) of 80-90 % of unfolded molecules with a time constant in the range 190-550 ps estimated b y linear extrapolation and model calculations. The remaining 10-20%. of the population folds to an off-pathway intermediate, 1, which is required to u nfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I <----> U <----> N). The slower of the two observa ble rates, which has a positive slope in the linear functional dependence o n the denaturant concentration indicating that an unfolding process under n ative-like conditions indeed exists, originates from the unfolding of I to U, which rate-limits the overall folding of these 10-20 % of molecules. Bot h fast and slow rates are independent of protein concentration and pH of th e refolding milieu, suggesting that the off-pathway intermediate is not a p rotein aggregate or trapped by heme misligation. The nature or type of unfo lded-state heme ligation does not interfere with refolding. Equilibrium pH titration of the unfolded state yielded coupled ionization of the two non-n ative histidine ligands, H26 and H33, with a pK(a) value of 5.85. A substan tial fraction of the unfolded population persists as the six-coordinate for m even at low pH, suggesting ligation of the two methionine residues, M65 a nd M80. These results have been used along with the known ligand-binding pr operties of unfolded cytochrome c to propose a model for heme ligation dyna mics. In contrast to refolding kinetics, the unfolding kinetics of reduced cytochrome c recorded by observation of Soret absorbance and tryptophan flu orescence are all slow, simple, and single-exponential. In the presence of 6.8 M GdnHCl, the unfolding time constant is similar to 300(+/- 125) ms. Th ere is no burst unfolding reaction. Simulations of the observed folding-unf olding kinetics by numerical solutions of the rate equations corresponding to the three-state I <----> U <----> N scheme have yielded the microscopic rate constants.