Georgiadis K E, Jhon N I, Einarsdóttir O
Department of Chemistry, University of California, Santa Cruz 95064.
Biochemistry. 1994 Aug 9;33(31):9245-56. doi: 10.1021/bi00197a028.
Intramolecular electron transfer and conformational changes in cytochrome c oxidase were studied at room temperature following the photodissociation of CO bound to mixed-valence enzyme (cytochrome a3(2+)-CO CuB+ cytochrome a3+ CuA2+) and fully reduced enzyme. Time-resolved optical absorption difference spectra were collected in the Soret region on time scales of nanoseconds to milliseconds using a gated optical spectrometric multichannel analyzer. A global exponential fitting procedure combined with a singular value decomposition method was used to analyze the transient difference spectra at various times following CO photolysis. The analysis shows that at least two processes, with apparent lifetimes of 1.4 microseconds and 11.1 ms, are present following the photodissociation of CO bound to the fully reduced enzyme. These are attributed to a conformational change and CO recombination at the cytochrome a3 site, respectively. Global analysis of the mixed-valence CO complex transient difference spectra showed the presence of five intermediates with apparent lifetimes of 1.0 microseconds, 5.2 microseconds, 83.7 microseconds, 10.5 ms, and 25.3 ms. The data on a microsecond time scale are consistent with a mechanism involving a conformational change at cytochrome a3, followed by electron transfer from cytochrome a3 to cytochrome a with subsequent electron transfer to CuA. One of the two processes on a millisecond time scale is attributed to CO recombination and the other to a structural rearrangement or heme-heme electron transfer. On the basis of this mechanism, the kinetics and the absorption spectra of the intermediates involved in the conformational and electron transfer dynamics of the mixed-valence enzyme were determined.
在室温下，研究了与混合价态酶（细胞色素a3(2 +)-CO CuB + 细胞色素a3 + CuA2 +）和完全还原酶结合的CO光解离后，细胞色素c氧化酶中的分子内电子转移和构象变化。使用门控光学光谱多通道分析仪，在纳秒至毫秒的时间尺度上，在索雷特区域收集时间分辨的光吸收差光谱。采用全局指数拟合程序结合奇异值分解方法，分析了CO光解后不同时间的瞬态差光谱。分析表明，与完全还原酶结合的CO光解离后，至少存在两个过程，其表观寿命分别为1.4微秒和11.1毫秒。这些分别归因于细胞色素a3位点的构象变化和CO重组。对混合价态CO复合物瞬态差光谱的全局分析表明，存在五个中间体，其表观寿命分别为1.0微秒、5.2微秒、83.7微秒、10.5毫秒和25.3毫秒。微秒时间尺度上的数据与一种机制一致，该机制涉及细胞色素a3的构象变化，随后电子从细胞色素a3转移到细胞色素a，随后再转移到CuA。毫秒时间尺度上的两个过程之一归因于CO重组，另一个归因于结构重排或血红素 - 血红素电子转移。基于该机制，确定了混合价态酶构象和电子转移动力学中涉及的中间体的动力学和吸收光谱。
