Kinetic Resolution of a Tryptophan-radical Intermediate in the Reaction Cycle of Paracoccus denitrificans Cytochrome c Oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa3 reacting with O2 between 83 μs and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of CuA, heme a, heme a3, and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). CuB and heme a3 were EPR silent during all stages of the reaction. CuA and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of CuA is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 μs and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a3 oxoferryl species PR (maxima at 430 nm and 606 nm) was completed in ∼130 μs, similar to the first oxidation phase of CuA and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from PR and decays to a hitherto undetected intermediate named FW*. FW* harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272*). The Trp-272* populates to 4-5% due to its relatively low rate of formation (t½ = 1.2 ms) and rapid rate of breakdown (t½ = 60 μs), which represents electron transfer from CuA/heme a to Trp-272*. The formation of the Trp-272* constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a3 oxoferryl species or to Cu 2+B. The potential role of Trp-272 in proton pumping is discussed.