Oxidation of ubiquinol by cytochrome bo(3) from Escherichia coli: Kinetics of electron and proton transfer
Oxidation of ubiquinol by cytochrome bo(3) from Escherichia coli: Kinetics of electron and proton transfer
复制标题
DOI：
10.1021/bi962478e
复制
发表时间：
1997-05-06
期刊：
影响因子：
2.9
通讯作者：
Brzezinski, P
中科院分区：
文献类型：
--
作者：
Ek, MS;Brzezinski, P
在这项研究中，我们使用所谓的流动闪光技术研究了细胞色素BO(3)与结合泛喹酚(QH(2))和氧气反应中的电子和质子转移。结果与线粒体细胞色素AA(3)的结果进行了比较。定性上，在两种酶中观察到了与电子转移相关的相同类型的吸光度变化，而质子化反应明显不同。在细菌QH(2)结合酶中，观察到三个动力学相，其时间常数分别类似于45目S、700目S和4ms的电子转移反应。第一阶段归因于氯化血红素b和o(3)的氧化以及“过氧基”中间体的形成。在加入除草剂HQNO后，没有观察到第二和第三相，HQNO取代了QH(2)的结合位置。它们分别归因于电子从QH(2)到血红素b和从血红素b到双核中心的转移。在这两种酶中，在最初的电子转移之后，每个酶分子(tau相当于90亩S)吸收0.9+/-0.3质子的速度较慢，这以前被认为是双核中心附近的一个基团的质子化。只有在细菌酶中，第二电子转移反应伴随着1.1+/-0.3H+的净释放，这归因于QH(2)氧化过程中的质子释放。随后，在第四电子转移到双核中心的过程中，1.2+/-0.4H+被较慢地吸收。在HQNO存在下，没有观察到两个最慢的质子化反应。
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.