Wiertz Frank G M, Richter Oliver-Matthias H, Ludwig Bernd, de Vries Simon
Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands.
J Biol Chem. 2007 Oct 26;282(43):31580-91. doi: 10.1074/jbc.M705520200. Epub 2007 Aug 30.
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 aa(3) reacting with O(2) between 83 micros 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 Cu(A), heme a, heme a(3), 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). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(). F(W)() 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 (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/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 a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
血红素 - 铜氧化酶的催化机制，即与质子泵浦耦合的电子转移，尚未完全明确。我们进行了微秒级冷冻超猝灭单周转实验，使用完全还原的细胞色素aa(3)在83微秒至6毫秒之间与O(2)反应。通过低温紫外可见光谱、X波段和Q波段电子顺磁共振光谱对捕获的中间体进行分析，从而能够确定Cu(A)、血红素a、血红素a(3)以及最近检测到的色氨酸自由基的氧化还原动力学（维尔茨，F.G.M.，里希特，O.M.H.，切列潘诺夫，A.V.，麦克米伦，F.，路德维希，B.，和德弗里斯，S.（2004年）《欧洲生物化学学会联合会快报》575，127 - 130）。在反应的所有阶段，Cu(B)和血红素a(3)通过电子顺磁共振光谱检测不到信号。Cu(A)和血红素a处于电子平衡状态，作为一个氧化还原对。Cu(A)的还原电位比血红素a低4.5毫伏。这两个氧化还原基团分两个阶段被氧化，表观半衰期分别为57微秒和1.2毫秒，在每个阶段共同向双核中心提供一个电子。血红素a(3)氧铁物种P(R)（在430纳米和606纳米处有最大值）的形成在大约130微秒内完成，类似于Cu(A)和血红素a的第一次氧化阶段。中间体F（在571纳米处吸光度最大）由P(R)形成，并衰变为一个迄今未检测到的名为F(W)()的中间体。F(W)()含有一个色氨酸自由基，通过Q波段电子顺磁共振光谱鉴定为严格保守的Trp - 272的色氨酸中性自由基（Trp - 272()）。由于其相对较低的形成速率（t((1/2)) = 1.2毫秒）和快速的分解速率（t((1/2)) = 60微秒），Trp - 272()的丰度达到4 - 5%，这代表了从Cu(A)/血红素a到Trp - 272()的电子转移。Trp - 272()的形成构成了催化循环的主要速率决定步骤。我们的研究结果表明，Trp - 272是一个氧化还原活性残基，在这方面与细胞色素c氧化酶的金属中心相当。Trp - 272是血红素a(3)氧铁物种或Cu(2 +)(B)的直接还原剂。文中还讨论了Trp - 272在质子泵浦中的潜在作用。
