Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach.
Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach.
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原位 bc(1) 复杂成分的光谱分析：超越传统的差分方法。
DOI：
10.1016/j.bbabio.2005.11.006
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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：
--
作者：
Shinkarev,VladimirP;Crofts,AntonyR;Wraight,ColinA
细胞色素 (cyt) bc1 复合物（泛醇：细胞色素 c 氧化还原酶）是线粒体和细菌电子传递链的中心酶。它含有丰富的假体基团，其中许多在可见光谱中具有显着但重叠的吸收带。 bc1复合物的细胞色素成分的动力学传统上是通过使用两个或多个不同波长处的吸光度变化的差异来跟踪的。这种差异波长 (DW) 方法已广泛用于球形红杆菌色素细胞中 bc1 复合物 Q 循环机制的开发和测试。然而，DW 方法并不能完全补偿来自其他成分的光谱干扰，这可能会严重扭曲振幅和动力学。 cyt bc1 转换的机制阐述需要一种克服这一限制的方法。在这里，我们将传统的 DW 方法与电子传输的最小二乘 (LS) 分析进行比较，该分析基于新确定的色素细胞中循环电子传输的所有单独组件的差异光谱。通过 LS 和 DW 方法分析了在不存在和存在特定抑制剂的情况下在不同波长下测量的多组动力学迹线。两种方法的比较表明，DW 方法不能充分校正成分之间的光谱重叠，并且当感兴趣成分的幅度变化很小时通常不可靠。特别是，它无法纠正对细胞色素 bL 的振幅和动力学的无关贡献。通过对发色成分（RC、ctot、bH 和 bL）的 LS 分析，我们表明，虽然 Q 循环模型仍然牢固地扎根，但有必要对各个成分的速率、幅度、延迟等进行定量重新评估。我们的结论是，进一步探索 bc1 复合体的机制，将需要 LS 反卷积来可靠地测量复合体各个成分的原位动力学。
The cytochrome (cyt) bc1complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc1complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc1complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc1turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt bL. From LS analysis of the chromophoric components (RC, ctot, bHand bL), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc1complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.