Abstract
The kinetics of formation of noncovalently bound ferrous complexes derived from fragments of horse heart cytochrome c have been investigated. When the reactions are initiated by combining ferrous heme fragments with an appropriate apofragment, in the presence of 50 mM imidazole, second order rate processes are observed with rate constants essentially the same as those reported with ferric heme fragments (Parr, G. R., and Taniuchi, H. (1979) J. Biol. Chem. 254, 4836-4842). An additional, probably consecutive, kinetic process is also demonstrated. If imidazole is not present in the reaction buffer, the kinetic profiles are dramatically altered. While this is partially due to aggregation (dimerization) of the ferrous heme fragments, it can nevertheless be demonstrated that the complementation reactions with apofragments are much faster than those observed with the corresponding ferric heme fragments (in the absence of imidazole). These results reflect the effect of the oxidation state of the heme iron on the folding mechanism and, thus, the manifold nature of protein folding pathways. The rate of reduction of productive ferric complexes by sodium ascorbate was investigated and biphasic reactions were found in all cases. The data indicate an equilibrium between two forms of the ferric complexes. The results of an experiment in which the complementation of ferric (1-25)H and (23-104) was carried out in the presence of sodium ascorbate indicate that the intermediate complex (Parr, G. R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 8914-8918) is not reducible by ascorbate. Thus, the increase in oxidation-reduction potential occurring on formation of the productive complex from the unbound heme fragment occurs at a late stage of the overall reaction, possibly coinciding with ligation of methionine 80 to the heme iron.
Highlights
From the Laboratory of Chemical Biology, National Institute ofiirthritis, Metabolism, and Digestive Diseases, National Institutes of Health,.Bethesda, Maryland20205 m e kinetics of formation of noncovalently bound hibit significant biological activity asmeasured by a variety of ferrous complexes derivedfromfragments of horse assay systems(2-7)
Inrelationtothe folding mechanism describedabove, we have investigated the kinetics of ious times and the absorbanceat 550 nm was recorded
Binding of Apofragments by Ferrous Heme FragrnentsBinding titrations wereperformed by adding aliquots ofa concentrated apofragment solution to a solution offerrous heme fragment (3.5 X IO-" M) in 0.1 M potassium phosphate, 25 mM sodium dithionite, pH 7.0,at approximately 25°C
Summary
Example, nonproductive complexes may be tightly bound (6, 7 ) and exhibit tryptophan fluorescence quenching and helical structure similar to the native protein(6, 7), but they lack an absorbance maximum at 695 nm in the ferric form (1,4, 6, 7), are not reducible by ascorbate (6, 7), have negative standard oxidation-reduction potentials (4), and adrevoid of biological activity(4, 6, 7). In order to determine the influence of Carbon Monoxide Binding-Solutions of complexes or free heme the valence of the heme iron on the folding pathway, studies fragments were prepared a t a concentration of approximately 2 X M in 0.1 M potassium phosphate,pH 7.0, containing 25 mM of the kinetics of formation of the complexes were extended sodium dithionite, and the absorbance at 550 nm was measured.
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