Radical in the Peroxide-Produced F-Type Ferryl Form of Bovine Cytochrome c Oxidase

The reduction of O(2) in respiratory cytochrome c oxidases (CcO) is associated with the generation of the transmembrane proton gradient by two mechanisms. In one of them, the proton pumping, two different types of the ferryl intermediates of the catalytic heme a(3)-Cu(B) center P and F forms, participate. Equivalent ferryl states can be also formed by the reaction of the oxidized CcO (O) with H(2)O(2). Interestingly, in acidic solutions a single molecule of H(2)O(2) can generate from the O an additional F-type ferryl form (F(•)) that should contain, in contrast to the catalytic F intermediate, a free radical at the heme a(3)-Cu(B) center. In this work, the formation and the endogenous decay of both the ferryl iron of heme a(3) and the radical in F(•) intermediate were examined by the combination of four experimental approaches, isothermal titration calorimetry, electron paramagnetic resonance, and electronic absorption spectroscopy together with the reduction of this form by the defined number of electrons. The results are consistent with the generation of radicals in F(•) form. However, the radical at the catalytic center is more rapidly quenched than the accompanying ferryl state of heme a(3), very likely by the intrinsic oxidation of the enzyme itself.