Oxidation of ubiquinol by cytochrome bo(3) from Escherichia coli: Kinetics of electron and proton transfer

In this study we have used the so-called flow-flash technique to investigate electron and proton transfer during the reaction between cytochrome bo(3) with bound ubiquinol (QH(2)) and dioxygen. The results are compared to those from the well-characterized mitochondrial cytochrome aa(3). Qualitatively, the same type of absorbance changes associated with electron transfer were observed in both enzymes whereas the protonation reactions were markedly different. In the bacterial QH(2)-bound enzyme, three kinetic phases with time constants of similar to 45 mu s, similar to 700 mu s, and similar to 4 ms associated with electron-transfer reactions were observed. The first phase is attributed to oxidation of hemes b and o(3) and formation of the ''peroxy'' intermediate. The second and third phases were not observed after addition of the herbicide HQNO, which displaces QH(2) from its binding site. They are attributed to electron transfer from QH(2) to heme b and from heme b to the binuclear center, respectively. In both enzymes, the initial electron transfer was followed by a slower uptake of 0.9 +/- 0.3 proton per enzyme molecule (tau congruent to 90 mu s), previously attributed to protonation of a group near the binuclear center. Only in the bacterial enzyme, the second electron-transfer reaction was accompanied by a net release of 1.1 +/- 0.3 H+, which is attributed to proton release during oxidation of QH(2). It was followed by a slower uptake of 1.2 +/- 0.4 H+ during transfer of the fourth electron to the binuclear center. The two slowest protonation reactions were not observed in the presence of HQNO.