Abstract
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.
Highlights
From the Institute of Hemoproteins, Department of Chemistry, State University of New York at Albany, Albany, New York 12222
Monomeric horse heart cytochrome c, used in these studies, was chromatographically prepared from a Sigma type 111 preparation according to published procedures. 2,3-Butanedione was purchased from Eastmanand/or Aldrich, and the monomeric form usedfor these studies was prepared by fractional distillation, the fraction between 86.5-88°C
The amino acid analysis of the solution after 22 and 44 h of reaction revealed a significant decrease in the arginine content from 2 residues [4] to 0.38 and 0.05 residues/molecule (Fig. 2), without anyindication of alteration of other aminoacids, including tryptophan and tyrosine
Summary
The mechanisms of the oxido-reduction reactions, including the possible locale of the site of attack, of a variety of organic and inorganic, nonphysiological molecules, have been determined during the last few years [4].Recently it was shown that reduction of the protein with ascorbate does not follow a classical inner sphere attack, i.e. through penetration into the heme crevice and sharing the coordination sphere of the ligands, nor does it follow an outer sphere, or remote, attack, but rather, an alternative path involving binding a t some molecular domain far removed from the opening of the crevice and migration of the electron through a rather low efficiencypathway [8].The possibility that thereduction site mayinvolve one of the two arginyl side chains has been arrived at primarily through the process of elimination. In thisreport we present further, more direct, evidence showing that the above suggestion is valid, for reactions with this nonphysiological donor
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