Fate of the excited triplet state of zinc cytochrome c in the presence of iron(III), iron(II), iron-free, and hems-free forms of cytochrome c

We study, by laser flash photolysis, the mechanism of quenching the triplet state of zinc cytochrome c, (3)Zncyt, by the iron(III), iron(II), iron-free, and heme-free forms of cytochrome c. The method of inserting zinc(II) ion into the heme group in a protein overcomes the difficulties in studying redox reactions between two heme proteins having similar absorption spectra. Quenching of positively charged (3)Zncyt by these positively charged reactants is promoted at high ionic strength, mu = 1500 mM, which shields electrostatic charge and weakens protein-protein repulsion. Upon addition of potential quenchers, the rate constant for (3)Zncyt decay becomes higher in the following order: ferrocytochrome c < iron-free cytochrome c much less than ferricytochrome c. However, upon addition of as much as 350 mu M heme-free cytochrome c, the rate of (3)Zncyt decay remains unchanged. The bimolecular rate constants for the reactions of (3)Zncyt are as follows: (1.5 +/- 0.2) x 10(4) M-1 s(-1) with ferrocytochrome c, (3.3 +/- 0.2) x 10(4) M-1 s(-1) with iron-free cytochrome c, and (5.1 +/- 0.3)x 10(6) M-1 s(-1) with ferricytochrome c. Oxidative quenching of (3)Zncyt by ferricytochrome c is proven by the observed formation of zinc cytochrome c cation radical. Energy transfer is responsible for the weak quenching of (3)Zncyt by iron-free cytochrome c and ferrocytochrome c, as evident in the lack of any observable transient radical-ions Zncyt(+) and Zncyt(-) and the good overlap of the emission band of 3Zncyt with the absorption bands of ferrocytochrome c and iron-free cytochrome c. (C) 2000 Elsevier Science S.A. All rights reserved.