The arginines of cytochrome c. The reduction-binding site for 2,3-butanedione and ascorbate.
The reactions of both ferric and ferrous horse heart cytochrome c with 2,3-butanedione, pH 7.5, 0.05 M bicarbonate buffer, under a variety of solution conditions, have been examined using amino acid integrity as the primary criterion. The only observable structural alteration was the modification of both the arginyl side chains. The reaction profiles were found to be dependent upon the presence or absence of borate and ascorbate. The single-phased modification of the arginyl side chains in ferrocytochrome c in the absence of borate is rendered biphasic in the presence of borate (borate/reagent ratio of 0.04) through substantial lowering of the rate of reaction of one of the two arginyl side chains. The addition of ascorbate inhibits the reaction in a competitive manner, particularly of the arginyl side chain undergoing rapid modification in its absence. The reactivity of both arginyl side chains to reagent for the ferricytochrome was appreciably larger than for ferrocytochrome c. The addition of reagent to ferricytochrome c also reduces heme iron, which is discerned from the development of a native-like spectrum in the region 500 to 600 nm. The differences in the reactivities of the arginyl side chains to 2,3-butanedione in the two valence states of heme iron are the reflection of small, but definite, conformational differences at the two sites in the two valence states of the protein. The concurrent reduction of heme iron and the modification of the arginyl side chains localizes the reduction and the reaction site for 2,3-butanedione. The inhibition by ascorbate of the reaction of one of the two arginines also identifies the binding domain. Of the two arginines, Arg-38 is suggested to be the ascorbate-binding site.
D000818:Animals; D001120:Arginine / Q000032:analysis*; D001205:Ascorbic Acid; D001665:Binding Sites; D002074:Butanones; D003574:Cytochrome c Group; D003931:Diacetyl; D006736:Horses; D009206:Myocardium; D010084:Oxidation-Reduction; D011485:Protein Binding; D013053:Spectrophotometry
D002074:Butanones; D003574:Cytochrome c Group; D001120:Arginine; D003931:Diacetyl; D001205:Ascorbic Acid