Cytochrome c: Ascorbate reduction site and possible electron‐transfer path

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.

SoxAX Cytochromes, a New Type of Heme Copper Protein Involved in Bacterial Energy Generation from Sulfur Compounds

“…The higher heme redox potential (E1) was found to be pH-independent in the range 6 Ͻ pH Ͻ 8, which is typical for His/Met-ligated cytochromes c. The observed E1 potential (ϩ183 Ϯ 10 mV, pH 7) is somewhat lower than that of analogues such as cytochrome c (horse heart, ϩ260 mV) (27). In contrast, the second potential (E2) was found to be extremely low (Ϫ479 Ϯ 10mV pH 7) ( Table 1).…”

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

“…At pH 7 the concentration of the true reductant, i.e. the ascorbate dianion, is very low (26,27) and therefore not expected to interfere with the ET events taking place between the partners of interest. Fig.…”

Kinetic studies on the reduction of cytochrome c. Reaction with dihydroxy conjugated compounds (catechols and quinols)

“…201 been extensively studied 19N9). Al-Ayash & Wilson (1979) suggested that dianionic forms of the reductant donate electrons to the exposed haem edge, but more recent work by Myer et al (1980) and provides evidence that ascorbate binds to the arginine-38 residue and that electron transfer to the haem proceeds via aromatic residues.…”