Menon A L, Hendrix H, Hutchins A, Verhagen M F, Adams M W
Department of Biochemistry and Molecular Biology, Center for Metalloenzyme Studies, University of Georgia, Athens 30602-7229, USA.
Biochemistry. 1998 Sep 15;37(37):12838-46. doi: 10.1021/bi980979p.
Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.
嗜热古菌激烈火球菌（Pf）中的丙酮酸铁氧化还原酶（POR）催化碳水化合物发酵的最后氧化步骤，即丙酮酸被氧化为乙酰辅酶A和二氧化碳，并伴随着铁氧化还原蛋白（Fd）的还原。POR由两个分子量约为120 kDa的“催化单元”组成。每个单元由四个亚基α、β、γ、δ组成，分子量分别约为44、36、20和12 kDa，并含有至少两个[4Fe-4S]簇。催化的精确机制以及各个亚基的作用尚不清楚。编码Pf POR δ亚基的基因已在大肠杆菌中表达，该蛋白在用铁和硫化物重构后进行了纯化。通过质谱测定，重构的δ亚基（recPOR-δ）为单体，分子量为11 879±1.2 Da，与基因序列预测的结果一致。纯化的recPOR-δ每摩尔含有8摩尔铁，根据其可见吸收特性判断，在85℃孵育2小时后仍保持完整。该蛋白的还原形式表现出两个自旋-自旋相互作用的[4Fe-4S]1+簇的特征性电子顺磁共振（EPR）谱。与还原型全酶的EPR特性相比，后者除了δ亚基内的两个[4Fe-4S]1+簇外，还含有第三个[4Fe-4S]1+簇。分离的recPOR-δ中两个4Fe簇在pH 8.0和24℃时的还原电位（-403±8 mV）随温度（-1.55 mV/℃）线性下降，直至82℃。RecPOR-δ替代了Pf Fd，作为Pf的两种氧化还原酶（POR和Fd:NADP氧化还原酶）的体外电子载体，并且POR全酶对其自身亚基（80℃时的表观Km = 1.0 μM）的表观亲和力高于对Fd（表观Km = 4.4 μM）。分离的δ亚基的分子和光谱性质以及氨基酸序列表明，它是由一种8Fe型Fd通过在N端添加约40个残基进化而来的，并且这种延伸使其能够与POR内的其他亚基相互作用。
