Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

Abstract

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H 2O 2 at pH 8.0 emit chemiluminescence with quantum yield Ф ⋍ 10 −9 and absolute maximum intensity I max ⋍ 10 5 hv/ s per cm 3 ( λ = 440), and exhibit exponential decay with a rate constant of 0.15 s −1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H 2O 2, a weak fluorescence in the region 400–620 nm with λ max = 465–510 nm ( λ exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan ( λ exc = 280 nm, λ em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ max = 530 nm ( λ exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen O 2( 1Δ g ) is not involved in either peroxidation or chemiluminescence. (6) The haem Fe 3+ group and H 2O 2 appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.