The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.