Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
Proc Natl Acad Sci U S A. 2010 Dec 7;107(49):21010-5. doi: 10.1073/pnas.1008603107. Epub 2010 Nov 19.
Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba(3) cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O(2)-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O(2)-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba(3) simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O(2) binding to heme a(3) at 90 μM O(2) occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 10(9) M(-1) s(-1) for O(2) binding to ba(3) in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O(2) bond cleavage in ba(3) of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O(2) to the active site in ba(3) and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a(3) site in ba(3), making the CO flow-flash method inapplicable.
使用 CO 流动闪光法研究细胞色素 c 氧化酶的动力学时，由于光解 CO 的去向，可能会影响实验结果。在这项时间分辨光吸收研究中，我们使用一种光不稳定的 O2 载体，比较了来自嗜热栖热菌的 ba(3)细胞色素 c 氧化酶在没有和有 CO 的情况下氧还原的动力学。引入了一种新的双激光激发方法，用 355nm 激光脉冲光解 O2 载体产生 O2，同时用第二束 532nm 激光脉冲同时光解完全还原的 CO 结合的 ba(3)。动力学分析揭示了一个顺序机制，在 90 μM O2 下，O2 分别与 heme a(3)结合的寿命为 9.3 和 110 μs，在没有和有 CO 的情况下，随后更快地断裂双氧键（4.8 μs），生成 P 中间物，同时氧化 heme b。在没有 CO 的情况下，ba(3)与 O2 的二级反应速率常数为 1×10(9) M(-1) s(-1)，比在有 CO 以及牛心酶中的观察值大 10 倍。ba(3)中 O2 键的断裂速度也比牛心酶快约 10 倍。这些结果表明，ba(3)和牛心酶中 O2 到达活性部位的可及性存在重要的结构差异，并且证明光解 CO 阻碍了 O2 进入 ba(3)的 heme a(3)位点，使得 CO 流动闪光法不适用。
