Weinberg Michael V, Jenney Francis E, Cui Xiaoyuan, Adams Michael W W
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
J Bacteriol. 2004 Dec;186(23):7888-95. doi: 10.1128/JB.186.23.7888-7895.2004.
Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 +/- 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85 degrees C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 micromol of rubredoxin oxidized/min/mg with apparent K(m) values of 35 and 70 microM for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 micromol of H(2)O(2) reduced/min/mg of rubrerythrin at 85 degrees C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.
在厌氧、还原条件下，通过多步色谱法从嗜热古菌激烈火球菌（Pyrococcus furiosus）中纯化出红素铁蛋白。它是一种同型二聚体，分子量为39.2 kDa，每个亚基含有2.9±0.2个铁原子。纯化后的蛋白质在85℃下具有过氧化物酶活性，以过氧化氢为底物，还原型激烈火球菌红素铁氧还蛋白作为电子供体。其比活性为36微摩尔红素铁氧还蛋白氧化/分钟/毫克，过氧化氢和红素铁氧还蛋白的表观K(m)值分别为35和70微摩尔。当红素铁蛋白与红素铁氧还蛋白及激烈火球菌NADH：红素铁氧还蛋白氧化还原酶结合时，完整系统以NADH作为电子供体来还原过氧化氢，在85℃下比活性为7.0微摩尔H(2)O(2)还原/分钟/毫克红素铁蛋白。奇怪的是，纯化后的（还原型）红素铁蛋白在被过氧化氢、空气或铁氰化物氧化时会沉淀。编码红素铁蛋白的基因（PF1283）在含有不同金属含量培养基中生长的大肠杆菌中表达。纯化后的重组蛋白每个亚基大约含有三个金属原子，范围从0.4个铁加2.2个锌到1.9个铁加1.2个锌，其中蛋白质的金属含量取决于大肠杆菌生长培养基的金属含量。重组形式的过氧化物酶活性与铁含量成正比。激烈火球菌红素铁蛋白是首个从嗜热菌或古菌中得到表征的此类蛋白，这些结果首次证明该蛋白在依赖NADH的过氧化氢：红素铁氧还蛋白氧化还原酶系统中发挥作用。红素铁蛋白被认为在最近定义的活性氧厌氧解毒途径中起作用。
