The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.
The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.
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DOI：
10.1016/s0021-9258(18)35891-5
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发表时间：
1992-11
期刊：
影响因子：
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通讯作者：
Gretchen L. AndersonSg;Jeffrey Williamsll;Russ HilleS
中科院分区：
文献类型：
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作者：
Gretchen L. AndersonSg;Jeffrey Williamsll;Russ HilleS
本文报道了粪产碱杆菌中亚砷酸盐氧化酶的纯化和初步性质。该酶由85 kDa的单体组成，含有一个钼，五个或六个铁和无机硫化物。在存在变性剂的情况下，亚砷酸盐氧化酶释放荧光物质，其光谱性质与由羟化酶类的含砷酶释放的蝶呤辅因子相同。从A. faecalis，每一个都是亚砷酸盐氧化酶的电子受体，并可能形成亚砷酸盐解毒的周质电子传递途径。亚砷酸盐氧化酶的完全还原需要3-4个还原当量，使用亚砷酸盐或连二亚硫酸盐作为电子源。低于20 K，氧化亚砷酸氧化酶表现出EPR信号与g值为2.03，2.01，和2.00，其整合到约0.4自旋/蛋白质。由于57 Fe的富集导致该EPR信号的加宽，因此产生该信号的中心必须含有铁。最可能的候选者是[4Fe-4S]高电位铁蛋白中心或[3Fe-4S]中心。在氧化的亚砷酸盐氧化酶中观察到的EPR信号在用亚砷酸盐或连二亚硫酸盐还原蛋白质后消失。同时，出现菱形EPR信号（g = 2.03，1.89，1.76），这是类似于Rieske型[2Fe-2S]集群和自旋量化为一个自旋/蛋白质。
The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.