Oxidation of ubiquinol by cytochrome bo3 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 bo3 with bound ubiquinol (QH2) and dioxygen. The results are compared to those from the well-characterized mitochondrial cytochrome alpha alpha3. 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 QH2-bound enzyme, three kinetic phases with time constants of approximately 45 micros, approximately 700 micros, and approximately 4 ms associated with electron-transfer reactions were observed. The first phase is attributed to oxidation of hemes b and o3 and formation of the "peroxy" intermediate. The second and third phases were not observed after addition of the herbicide HQNO, which displaces QH2 from its binding site. They are attributed to electron transfer from QH2 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 approximately 90 micros), 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 QH2. 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.
Svensson Ek
M
M
Department of Biochemistry and Biophysics, University of Göteborg, Chalmers University of Technology, Sweden.
Brzezinski
P
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011522:Protons; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines; D011522:Protons; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; C003741:ubiquinol; D010100:Oxygen