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,

No abstract

No abstract

The ascorbate reduction reaction of the native and urea-perturbed forms, 0-8M urea, of horse heart ferricytochrome c is found to be a three-step process: a urea-dependent equilibrium step between a reducible and an irreducible form with a midconcentration of urea of 7.4M, a binding step with a binding constant of 5.9M-', and a reduction step with a urea-independent rate constant of 2.9 f 0.3 s-I [J. Biol. Chem. 255,9666 (1980)l. The effect of adding urea, in addition to the generation of an irreducil$e form, is a slight lowering of the ascorbate-protein binding constant, 5.9 to 2.7M-I, which is limited to the 0-5.5M concentration range. The thermodynamics of the ureadenaturation process also yields a three-step mechanism, N-Xl-X2-D, with midconcentrations of urea of 2.5-3M, 6.2M, and 7 S M , respectively, where N, D, and the X , are the native, the 9-M-urea, and the intermediate forms. The three processes are described as the loosening of the heme crevice opening, the solvent exposure of the polypeptide backbone, and the disruption of the tryptophan-porphyrin interactions, respectively [Biochemistry 19, 199 (1980)l. The reaction of the protein with 2,3-butanedione, a group-specific reagent for the guanidinium groups and an electron donor for this protein, is inhibited in the presence of ascorbate, but only one of the two functional groups is involved [J. Biol. Chem. 255, 11094 (1980)l. A correlation of kinetic and thermodynamic observations led to the conclusion that the ascorbate reduction of the protein is independent of the state of the heme crevice opening and of the polypeptide organized structures; instead, it is determined by the integrity of the tryptophan indole-porphyrin interactions. This information, when taken in conjunction with the selective inhibition of the reaction of the arginine side chains by ascorbate, establishes the binding site of ascorbate as one of the two arginyl side chains, and not the opening of the crevice or its vicinity. From the three-dimensional structure of the protein, and taking into consideration the variability of the protein sequence, it is suggested that Arg-38 is the ascorbate binding site, and that the electronic interaction between the indole of Trp-59 and the porphyrin moiety must constitute, at least in part, the electron-transfer path to heme iron.

No abstract