Thomas M A, Gervais M, Favaudon V, Valat P
Eur J Biochem. 1983 Oct 3;135(3):577-81. doi: 10.1111/j.1432-1033.1983.tb07691.x.
The reversible association of the Zn2+-substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate-reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn-cytochrome c complexing to defined proteolytic fragments of flavocytochrome b2, either heme-b2-containing monomers or a flavin-linked tetramer. All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2. These data allowed localization of the single high-affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers. Quenching of the Zn-porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b2 group.
通过荧光分析法研究了锌离子取代的异常汉逊酵母细胞色素c二聚体（Thomas等人，本期前文）与氧化态或乳酸还原态的黄素细胞色素b2的可逆结合。相同的方法已用于测定锌细胞色素c与黄素细胞色素b2特定蛋白水解片段的络合，这些片段包括含血红素b2的单体或黄素连接的四聚体。除了分离出的细胞色素b2核心外，所有这些片段都显示出与天然黄素细胞色素b2相似的结合化学计量、解离常数（Kd）值和离子强度依赖性。这些数据使得能够将细胞色素c的单个高亲和力结合位点定位在黄素细胞色素b2原体脱氢酶结构域中的一个特定球体上。各种复合物中锌卟啉c荧光的猝灭仅伴随着荧光寿命的微小变化，并且与血红素b2基团的存在或氧化还原状态没有任何直接关系。
