The glutathione free radical equilibrium, GS . + GS −⇌ GSS .−G, mediating electron transfer to FE(III) -cytochrome c

Abstract

The GS .+GS − ⇌ GSS .− G equilibrium (1) was reinvestigated as a function of pH and ionic strength, using pulse radiolysis to oxidize GSH to GS .. All radicals formed by water radiolysis can be converted to populate equilibrium (1), which presents an interesting chemical junction between an electron acceptor (GS .) and an electron donor (GSS .−G). The secondary decay of the GS ./GSS .− G couple into the reducing carbon-centred radical G(C α .)SH, as observed in alkaline solution [10] (Grierson et al., Int. J. Radiat. Biol. 62 (1992) 265), seems to be of minor importance in neutral solution. Reduction of Fe(III)-cytochrome c at pH 6.8, after pulse radiolytic generation of the GS ./GSS .− G couple in absence of oxygen, proceeds with half-lifes in the order of 100 to 200 μs. From the concentration dependent rate and efficiency of reduction it is concluded that GSS .− G is the reducing entity, k(GSS .− G + Fe(III)Cyt c) ≈ 6 x 1 0 7 M −1 s −1. The efficiency of reduction reaches about 72% (of GS .) for γ-radiolysis of deaerated solutions containing GSH and Fe(III)-cytochrome c at pH 6.8, even though the reverse reaction (-1) is favoured at this pH (where only few % of GS . equilibrate to GSS .−G). Reduction is less efficient under pulse radiolysis conditions due to competing radical-radical termination (e.g. GS .+ GS .-→ GSSG). In presence of oxygen the efficiency of reduction is even higher, 95% for γ-radiolysis, and the rate of reduction indicates that O 2 .− is the reluctant. Reversible formation of thiyl peroxy radicals (GS .+ O 2 ⇌ GSOO .) seems to be overruled, via equilibrium (1), by irreversible electron transfer from GSS .− G to O 2, for which a rate constant of 5.1 x 10 8 M −1 s −1 was estimated. The kinetics of copper-catalyzed reduction of Fe(III)-cytochrome c by GSH were investigated by stopped-flow techniques. The results presented indicate that the Cu(I)-thiolate complex is the reducing entity. It is concluded that Cu(II) does not interact with GSH to form the unbound GS . radical, and that reduction in this case is not mediated by equilibrium (1).