Critical analysis of the extinction coefficient of chloroplast cytochrome f.
Critical analysis of the extinction coefficient of chloroplast cytochrome f.
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叶绿体细胞色素f消光系数的批判性分析。
DOI：
10.1016/s0005-2728(96)00164-8
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Whitmarsh,J
中科院分区：
文献类型：
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作者：
Metzger,SU;Cramer,WA;Whitmarsh,J
在产氧光合作用中，细胞色素bf复合物连接光系统II和光系统I之间的电子传递。复合物的最大亚基是细胞色素f，一种32 kDa的多肽，通过位于羧基末端附近的跨膜α螺旋锚定在膜上。用X射线晶体学方法测定了芜菁细胞色素f可溶性结构域的三维结构，分辨率为1.96 μ m。与先前解决的可溶性c型细胞色素结构相比，该结构揭示了几个新的特征，包括主要的β-链基序，酪氨酰残基的N-末端α-氨基作为正交配体，以及结合的内部水链。在这里，我们报告了一个新的和前所未有的消光系数细胞色素f。使用吡啶血色素测定，对于相对于等吸光度波长在554 nm处的α带波长峰，芜菁细胞色素f的可溶性结构域的还原-氧化消光系数为26±1 mM−1cm− 1（534，543.5和560.5 nm），菠菜细胞色素f相对于等吸光波长（533.5，543.3和560.2）为25±1 mM− 1 cm − 1。这里报告的消光系数显着高于以前公布的细胞色素f的值。我们认为早期的测定低估了细胞色素f的消光系数，这可能也适用于常用的细胞色素b6的消光系数。与大多数其他C型细胞色素相比，细胞色素f消光系数较大，这可能是由于细胞色素f血红素的独特轴向配体。在pH7.5时，可溶性芜菁细胞色素f的电位为362±5 mV。中点电位在5.0 ~ 8.5范围内与pH无关，在8.5 ~ 10.5范围内与pH相关（−58 mV/pH单位），氧化形式的pK接近9。将一些纯化的芜菁和菠菜细胞色素f样品在−20°C下储存，改变了一部分蛋白质中的血红素环境，使中点电位移到−165 mV（pH 7.5）附近，α带吸收光谱的峰值从554 nm移到552 nm。
In oxygenic photosynthesis the cytochrome bf complex links electron transport between photosystem II and photosystem I. The largest subunit of the complex is cytochrome f, a 32-kDa polypeptide that is anchored in the membrane by a transmembrane alpha helix located near the carboxyl end. The three-dimensional structure of the soluble domain of cytochrome f isolated from turnip has been determined by X-ray crystallography to 1.96 Å resolution. The structure revealed several novel features compared to previously solved soluble c-type cytochrome structures, including a predominant β-strand motif, the N-terminal α-amino group of a tyrosyl residue as an orthogonal ligand, and a bound internal water chain. Here we report a novel and unprecedented extinction coefficient for cytochrome f. Using the pyridine hemochrome assay, the reduced minus oxidized extinction coefficient for the soluble domain of turnip cytochrome f was 26±1 mM−1cm−1for the α-band wavelength peak at 554 nm relative to the isosbestic wavelengths (534, 543.5 and 560.5 nm), and 25±1 mM−1cm−1for spinach cytochrome f relative to the isosbestic wavelengths (533.5, 543.3 and 560.2). The extinction coefficients reported here are significantly higher than previously published values for cytochrome f. We believe earlier determinations underestimated the cytochrome f extinction coefficient and that the same is likely true for commonly used extinction coefficients of cytochrome b6. The cytochrome f extinction coefficient is large compared to most other c-type cytochromes, which could be due to the unique axial ligand of the cytochrome f heme. Polarographic measurements show the midpoint potential of soluble turnip cytochrome f to be 362±5 mV at pH 7.5. The midpoint potential was pH-independent from 5.0 to 8.5, and pH-dependent from pH 8.5 to 10.5 (−58 mV/pH unit) with a pK on the oxidized form near 9. Storage of some samples of purified turnip and spinach cytochrome f at −20°C modified the heme environment in a fraction of the protein, shifting the midpoint potential to near −165 mV (pH 7.5) and the peak of the α-band absorption spectrum from 554 nm to 552 nm.