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Equilibrium studies of the urea denaturation of horse heart ferricytochrome c, pH 7.0, through alteration of the absolute extinction of the 695-nm band and of the fluorescence efficiency of the tryptophan side chain, have been reported. The denaturation profiles using the two probes have been analyzed in terms of similarities and differences as well as to determine the nature of the intermediate forms. The intermediate forms in the 4-4.5 M urea concentration range have been further characterized by circular dichroism spectroscopy in the Soret and the intrinsic absorption regions, stability to temperature and pH, and reactivity of the methionine and histidine side chains to bromoacetic acid. The scheme N + XI + X2 D, in which N and D are the 0 and 9 M urea forms and XI and X2 are the two intermediate forms,with midconcentrations of urea of 2-2.5, 6.2, and 6.9 M, respectively, for the three transitions, is proposed as an explanation of the observed denaturation profiles of the protein.The N to XI transition is seen in the enhanced absorptivity of the 695-nm band and the further quenching of tryptophan fluorescence. Form X, exhibits lowered temperature and pH stability, a nativelike intrinsic CD spectrum, and reactivity of both His-18 and Met-80 to bromoacetic acid. The X I to X2transition is apparent only in the 695-nm absorptivity-urea profile, while the X2 to D transition is discerned from both the enhancement of tryptophan fluorescence and the final quenching of the 695-nm absorptivity. The extent of alteration T e structural, conformational, and possibly functional aspects of enzymes can be better understood from a detailing of the folding and unfolding processes. In the case of the constituents of the electron-transport chain, cytochrome c is the only component susceptible to such investigation, primarily because of its availability in a pure, well-defined form and the wealth of information regarding its structure,

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