Oxidation-state-dependent reactions of cytochrome c with the trioxidocarbonate(•1−) radical: a pulse radiolysis study

Abstract

The reaction of the trioxidocarbonate(*1-) radical (CO (3) (*-) , "carbonate radical anion") with cytochrome c was studied by pulse radiolysis at alkaline pH and room temperature. With iron(III) cytochrome c, CO (3) (*-) reacts with the protein moiety with rate constants of (5.1 +/- 0.6) x 10(7) M(-1) s(-1) (pH 8.4, I approximately 0.27 M) and (1.0 +/- 0.2) x 10(8) M(-1) s(-1) (pH 10, I = 0.5 M). The absorption spectrum of the haem moiety was not changed, thus, amino acid radicals produced on the protein do not reduce the haem. The pH-dependent difference in rate constants may be attributed to differences in ionization states of amino acids and to the change in the conformation of the protein. With iron(II) cytochrome c, CO (3) (*-) oxidizes the haem quantitatively, presumably via electrostatic guidance of the radical to the solvent-accessible haem edge, with a different pH dependence: at pH 8.4, the rate constant is (1.1 +/- 0.1) x 10(9) M(-1) s(-1) and, at pH 10, (7.6 +/- 0.6) x 10(8) M(-1) s(-1). We propose that CO (3) (*-) oxidizes the iron center directly, and that the lower rate observed at pH 10 is due to the different charge distribution of iron(II) cytochrome c.

Reactions of heme proteins with carbonate radical anion

“…In the case of cytochrome c (in its ferric form), we confirmed the results obtained earlier by Domazou and Koppenol [8]. It has also been shown that CO 3…”

“…•-measured at pH 10 agrees perfectly with that observed by Domazou and Koppenol [8]. However, the same authors have found that the value of the rate constant of this reaction at pH 8.4 is lower and equals 5.1 9 10 7 M -1 s -1 .…”

“…It seems reasonable that the mismatch between our results for cytochrome c and those obtained by Domazou and Koppenol [8] may be due to the higher ionic strength of their solutions (0.25 M bicarbonate).…”

“…•-with catalase, HRP, and cytochrome c. The values obtained here for cytochrome c (at pH 8 and 10) were compared with those published earlier [8].…”

“…Thus, CO 3 •-radicals can damage proteins and nucleic acids. Recently, it has been reported that CO 3 •-directly oxidizes the Fe(II) heme center of cytochrome c [8]. It has also been found that, in the case of the reaction of oxyhemoglobin and oxymyoglobin with CO 3 •-, the initially formed amino acid radicals oxidize the iron center [9,10].…”