Pieulle L, Guigliarelli B, Asso M, Dole F, Bernadac A, Hatchikian E C
Unité de Bioénergétique et Ingénierie des Protéines, CNRS, Marseille, France.
Biochim Biophys Acta. 1995 Jul 3;1250(1):49-59. doi: 10.1016/0167-4838(95)00029-t.
We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.
我们报道了从硫酸盐还原菌非洲脱硫弧菌中首次纯化和鉴定丙酮酸-铁氧化还原蛋白氧化还原酶（POR）。分离得到的该酶在有氧条件下高度稳定，比活性为14 U/mg。非洲脱硫弧菌POR是一种256 kDa的同型二聚体，含有硫胺焦磷酸（TPP）和铁硫簇。对该酶的电子顺磁共振光谱研究表明，每个亚基存在三个[4Fe-4S]2+/1-中心。这三个中心的中点电位分别为-390 mV、-515 mV和-540 mV。POR的催化机制涉及一个自由基中间体，当加入辅酶A时该中间体消失。根据从TPP到受体的电子传递链对这种行为进行了讨论。与其他嗜温菌POR相比，经二硫赤藓糖醇激活的该酶表现出异常高的活性，且与激活前的酶相比，对氧气变得非常敏感。对分离得到的酶和激活后的酶的电子顺磁共振光谱比较表明，激活过程既不影响FeS中心的性质，也不影响其排列。非洲脱硫弧菌铁氧化还原蛋白I和II作为该酶的生理电子载体。通过免疫金标记显示POR位于细胞质中。
