Reduction of Ferricytochrome c by Dithionite Ion: Electron Transfer by Parallel Adjacent and Remote Pathways

Abstract

The kinetics of the reduction of horseheart ferricytochrome c by sodium dithionite (phosphate buffer-sodium chloride; pH 6.5, ; = 1.0, 250) features two reaction pathways; one with the rate constant kg = 1.17 X 104 M-l sec , the other with the rate constant klk2/k_,-6.0 X 104 M-1 sec-. These pathways are interpreted in terms of remote attack (possibly by way of the exposed edge of the porphyrin system) and adjacent attack (requiring the opening of the heme crevice). The limiting rate for the adjacent pathway (k, = 30 sec-) is in good agreement with the rate of heme-crevice opening of ferricytochrome c determined in other studies. The implication of the adjacent attack pathway to the function of cytochrome c in vivo is discussed.Cytochrome c, an electron carrier in the mitochondrial respiratory chain, is a relatively stable metalloprotein consisting of one heme group and one polypeptide chain. The heme group is covalently bonded to the protein by thioether bridges between the porphyrin ring and two cysteine residues in the peptide chain; in ferricytochrome c (Fe"i'cyt c) the heme group lies in a crevice of the essentially globular protein with an edge of the porphyrin ring located at the surface of the molecule (1). The iron atom is situated in the plane of the porphyrin ring with its fifth and sixth coordination sites occupied by a ring nitrogen atom of histidine-18 and the sulfur atom of methionine-80.Recent studies of substitution (2) and electron transfer (3) reactions of ferricytochrome c have revealed two pathways for electron transfer: an adjacent attack pathway requiring the rupture of the iron-sulfur bond or the opening of the heme crevice (with a rate constant of 60 sec at 25°), and a remote attack pathway involving an indirect route, possibly the exposed edge of the porphyrin ring system. This paper is concerned with the reaction of ferricytochrome c with the potent, frequently used reducing agent, dithionite [E' for the reduction of sulfur(IV) to dithionite is -0.46 v at pH 7 (4)]. This work extends the earlier studies (3), and shows that the mechanism of reduction of ferricytochrome c may depend upon both the nature of the reducing agent and its concentration. MATERIALS AND METHODSSolutions 5-10 /M in Sigma horse-heart Type III cytochrome c (normally used without further purification) were prepared in 0.04 M phosphate-0.91 M NaCl, having a pH of 6.5. Cytochrome c, purified in the following manner, was used in a Abbreviations: Fe"' cyt c, ferricytochrome c; Fe"l cyt c, ferrocytochrome c. * This is publication III in a series, "Mechanisms of the Reactions of Cytochrome c." The preceding paper in this series is ref. 3.t Author to whom reprint requests should be addressed. 1701 few runs. A 60-mg sample of the commercial hemeprotein was subjected to gel filtration on a 2 X 20-cm column of Sephadex G-50 (fine) in 0.04 M phosphate (pH 6.5)-0.91 M NaCl at 40. The center fraction (about 3 ml) of the filtrate was loaded onto a 4 X 20-cm column of Bio-Rex 70 (100-200 mesh, Na+ form) and eluted with a li...

The reaction of ferric cytochrome c with dithionite at very low ionic strength

“…At very low I, it is apparent that the fast phase of the reaction features different mechanisms at high and low dithionite concentrations, similarly to the behaviour at very high ionic strength (1 N NaCl) [2] . Creutz and.Sutin [2] suggested two different bimolecular pathways for the fast reduction by dithionite: one, a direct remote attack; the other, an adjacent pathway requiring the opening of the heme crevice. We have evaluated the limiting rate constants for the opening of the heme crevice as a function of salt concentration (table 1).…”

“…It is quite obvious that the reduction studied at very high ionic strength (1 N NaCl) at neutral pH [2] is formed ( fig.1, fig.3); this was not observed during was monophasic, because such conditions permit the the reduction by ascorbate. This difference results existence of only one ferric cytochrome c conformer, from the much faster kinetics observed for dithionite.…”

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Sodium dithionite stock solution of 100 mM was first prepared in 1 M tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 9.0) under argon, and then dilute solutions were made and used within a few hours. The concentrations of all the dithionite solutions were standardized at 420 nm (e=1.02 mM À1 cm À1 ) [48]. Addition of sodium dithionite resulted in a decrease of the 490-nm feature (or of the 502-nm feature in the case of desulforedoxin) of the spectrum, characteristic of the ferric form of the protein.…”

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

“…A 100 mM sodium dithionite stock solution was first prepared in Tris-HCl (pH 9.0) under argon, and then diluted solutions were made and used within a few hours. All the dithionite solutions concentrations were standardized at 420 nm (e=1.02 mM À1 cm À1 ) [38]. Addition of sodium dithionite resulted in a decrease of the 490-nm feature of the spectrum, characteristic of the Fe 3+ form of rubredoxin.…”