Spectral and kinetic resolution of the bc1 complex components in situ: a simple and robust alternative to the traditional difference wavelength approach.
Spectral and kinetic resolution of the bc1 complex components in situ: a simple and robust alternative to the traditional difference wavelength approach.
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bc1 复杂组分的原位光谱和动力学分辨率：传统差异波长方法的简单而稳健的替代方案。
DOI：
10.1016/j.bbabio.2006.04.003
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Wraight,ColinA
中科院分区：
文献类型：
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作者：
Shinkarev,VladimirP;Crofts,AntonyR;Wraight,ColinA
bc1 复合物（泛醇：细胞色素 c 氧化还原酶，复合物 III）的细胞色素 (cyt) 成分的动力学传统上是通过使用两个或多个不同波长下吸光度变化的差异来跟踪的。然而，这种差分波长 (DW) 方法在分离具有重叠光谱带的成分的吸光度变化方面精度有限。为了解析球形红细菌色素细胞中各个成分的动力学，我们基于最少数量的不同波长的测量，测试了最小二乘 (LS) 分析的简化版本。简化 LS 分析的成功很大程度上取决于套件中使用的波长。 “传统”组 6 个波长（542、551、561、566、569 和 575 nm）通常用于 DW 方法来表征色素细胞中 cyt ctot(cyt c1+cyt c2)、cyt bL、cyt bH 和 P870 的动力学，也可用于通过简化的 LS 分析来确定这些成分，其中提高了各个组件的分辨率。然而，当需要有关细胞色素c1和c2的信息时，该集合是不够的。我们确定了多组可选的 5 和 6 波长，可用于同时确定所有 5 种成分（P870 和细胞色素 c1、c2、bL 和 bH）的动力学，其精度与基于全套波长（1 nm 间隔）的 LS 分析相当。我们得出的结论是，基于少量精心选择的波长的 LS 反卷积的简化版本比传统的 DW 方法提供了稳健且显着的改进，因为它考虑了不同成分的光谱干扰，并且在需要有关所有五个单独成分的信息时使用更少的测量。使用简化和完整的 LS 分析，我们测量了 bc1 复合物的所有细胞色素成分在特定抑制剂不存在和存在的情况下的同时动力学，发现它们与修改后的 Q 循环的预期吻合良好。这是第一项同时测量原位功能的 bc1 复合物的所有细胞色素和反应中心成分的动力学的研究，并在较长的时间范围内进行完全解卷积。
The kinetics of the cytochrome (cyt) components of the bc1complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt ctot(cyt c1+cyt c2), cyt bL, cyt bH, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c1and c2is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c1, c2, bL, and bH) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc1complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc1complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.