AMIDE PROTON-EXCHANGE RATES OF OXIDIZED AND REDUCED SACCHAROMYCES-CEREVISIAE ISO-1-CYTOCHROME-C

Proton NMR spectroscopy was used to determine the rate constant, k(obs ), for exchange of labile protons in both oxidized (Fe(III)) and reduc ed (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbon e amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular seconda ry structure as well as in OMEGA-loops. Furthermore, there is no corre lation between k(obs) and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consist ent with the local unfolding model. Comparisons of the free energy cha nge for denaturation, DELTAG(d), at 298 K to the free energy change fo r local unfolding, DELTAG(op), at 298 K for the oxidized protein sugge st that certain conformations possessing higher free energy than the d enatured state are detected at equilibrium. Reduction of the protein r esults in a general increase in DELTAG(op). Comparisons of DELTAG(d) t o DELTAG(op) for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatu red form. This persistent structure in high-energy conformations of th e reduced form appears to involve the axially coordinated heme.