TY - JOUR
T1 - Simultaneous reduction of iron-sulfur protein and cytochrome bL during ubiquinol oxidation in cytochrome bc1 complex
AU - Zhu, Jian
AU - Egawa, Tsuyoshi
AU - Yen, Syun Ru
AU - Yu, Linda
AU - Yu, Chang An
PY - 2007/3/20
Y1 - 2007/3/20
N2 - The key step of the protonmotive Q-cycle mechanism of the cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. It was postulated that the iron-sulfur protein (ISP) accepts the first electron from ubiquinol to generate ubisemiquinone anion to reduce bL. Because of the difficulty of following the reduction of ISP optically, direct evidence for the early involvement of ISP in ubiquinol oxidation is not available. Using the ultra-fast microfluidic mixer and the freeze-quenching device, coupled with EPR, we have been able to determine the presteady-state kinetics of ISP and cytochrome bL reduction by ubiquinol. The first-phase reduction of ISP starts as early as 100 μs with a t1/2 of 250 μs. A similar reduction kinetic is also observed for cytochrome bL, indicating a simultaneous reduction of both ISP and bL. These results are consistent with the fact that no ubisemiquinone was detected at the Q p site during oxidation of ubiquinol. Under the same conditions, by using stopped flow, the reduction rates of cytochromes bH and c 1 were 403 s-1 (t1/2 1.7 ms) and 164 s -1 (t1/2 4.2 ms), respectively.
AB - The key step of the protonmotive Q-cycle mechanism of the cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. It was postulated that the iron-sulfur protein (ISP) accepts the first electron from ubiquinol to generate ubisemiquinone anion to reduce bL. Because of the difficulty of following the reduction of ISP optically, direct evidence for the early involvement of ISP in ubiquinol oxidation is not available. Using the ultra-fast microfluidic mixer and the freeze-quenching device, coupled with EPR, we have been able to determine the presteady-state kinetics of ISP and cytochrome bL reduction by ubiquinol. The first-phase reduction of ISP starts as early as 100 μs with a t1/2 of 250 μs. A similar reduction kinetic is also observed for cytochrome bL, indicating a simultaneous reduction of both ISP and bL. These results are consistent with the fact that no ubisemiquinone was detected at the Q p site during oxidation of ubiquinol. Under the same conditions, by using stopped flow, the reduction rates of cytochromes bH and c 1 were 403 s-1 (t1/2 1.7 ms) and 164 s -1 (t1/2 4.2 ms), respectively.
KW - EPR
KW - Electron transfer
KW - Rapid freeze-quenching
KW - Reductase
KW - Stopped-flow
KW - Ubiquinol-cytochrome c
UR - http://www.scopus.com/inward/record.url?scp=34247569891&partnerID=8YFLogxK
U2 - 10.1073/pnas.0607812104
DO - 10.1073/pnas.0607812104
M3 - Article
C2 - 17360398
AN - SCOPUS:34247569891
SN - 0027-8424
VL - 104
SP - 4864
EP - 4869
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 12
ER -