Agalidis I, Othman S, Boussac A, Reiss-Husson F, Desbois A
Centre de Génétique Moléculaire, CNRS 91198, Gif-sur-Yvette, France.
Eur J Biochem. 1999 Apr;261(1):325-36. doi: 10.1046/j.1432-1327.1999.00277.x.
The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.
嗜胶红假单胞菌反应中心的四血红素细胞色素c亚基在存在辛基β-D-硫代葡萄糖苷的情况下，通过硫酸铵沉淀并在pH 9的Deriphat 160溶液中溶解进行分离。对该纯化细胞色素的几种生化特性进行了表征。特别是，它形成小的寡聚体，其N端氨基酸被封闭。在存在或不存在二氨基杜烯、抗坏血酸和连二亚硫酸盐的情况下，通过吸收光谱、电子顺磁共振（EPR）光谱和共振拉曼光谱研究了分离细胞色素的不同氧化/还原状态。所有数据表明，两个血红素可被抗坏血酸快速还原，一个血红素可被抗坏血酸缓慢还原，一个血红素仅可被连二亚硫酸盐还原。抗坏血酸快速还原的血红素具有与反应中心结合细胞色素的两个低电位血红素非常相似的顺磁性质（gz = 3.34），但其α带在还原状态下分裂为两个成分，分别在552 nm和554 nm处达到峰值。共振拉曼光谱表明，它们的轴向配体不变，为His/Met和His/His。抗坏血酸缓慢还原的血红素和连二亚硫酸盐还原的血红素被归为结合细胞色素的两个高电位血红素。它们的α带在551 nm处发生蓝移，gz值降至2.96，尽管轴向配体（His/Met）保持不变。得出的结论是，这些血红素估计300 mV的电位降反映了它们溶剂可及性的变化，而gz的降低表明其配位球对称性增加。这些结构修饰损害了细胞色素作为反应中心光氧化细菌叶绿素二聚体电子供体的基本功能。与天然状态相比，分离的细胞色素无法有效还原从缺失四血红素的嗜胶红假单胞菌突变体中纯化的反应中心。尽管细胞色素发生了构象变化，但它的四个血红素仍分为两组，一组为一对低电位血红素，另一组为一对高电位血红素。
