Shinkarev Vladimir P, Crofts Antony R, Wraight Colin A
Department of Biochemistry, University of Illinois, 156 Davenport Hall, 607 South Mathews Avenue, Urbana, Illinois 6l80l, USA.
Biochemistry. 2006 Jun 27;45(25):7897-903. doi: 10.1021/bi060172u.
In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.
在球形红细菌的载色体中，细胞色素（cyt）c(1)和c(2)具有高度重叠的光谱，对它们进行光谱解卷积是一项具有挑战性的任务。因此，对紫色细菌中bc(1)复合物不同细胞色素组分的动力学分析通常仅报告细胞色素c(1)+细胞色素c(2)的总动力学。在这里，我们利用新测定的各个组分的差光谱，通过最小二乘法（LS）解卷积原位解析细胞色素c(1)和c(2)的动力学。我们发现，基于每个组分特有的两个不同波长处吸光度的差异，细胞色素c(1)和c(2)的动力学与使用传统差异波长（DW）方法测得的动力学有显著差异。特别是，使用先前推荐的波长对时，仪器校准的差异导致用DW方法测得的闪光诱导细胞色素c(1)氧化动力学比LS方法测定的更快（在存在抗霉素的情况下，半衰期约为120微秒，而LS方法约为235微秒）。此外，LS方法揭示了细胞色素c(1)氧化动力学中约50微秒的延迟，使用DW方法时这一延迟被掩盖了。我们将这一延迟归因于光合反应中心光激活后导致细胞色素c(1)氧化的所有过程，尤其是细胞色素c(2)从反应中心的解离。我们还发现，由于b型血红素变化引起的光谱污染，在时间长于1毫秒时，用DW方法测得的细胞色素c(1)和c(2)的动力学都会显著失真。细胞色素c(1)和c(2)的成功光谱解卷积以及在动力学分析中纳入这两种细胞色素，显著增加了用于原位深入理解bc(1)周转机制的数据。
