Anderson G L, Williams J, Hille R
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
J Biol Chem. 1992 Nov 25;267(33):23674-82.
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.
本文描述了从粪产碱杆菌中纯化亚砷酸盐氧化酶并对其进行初步表征的过程。该酶由一个85 kDa的单体组成，含有一个钼原子、五个或六个铁原子以及无机硫化物。在变性剂存在的情况下，亚砷酸盐氧化酶会释放出一种荧光物质，其光谱特性与含钼羟化酶类释放的蝶呤辅因子相同。从粪产碱杆菌中分离出的天青蛋白和一种c型细胞色素，均可作为亚砷酸盐氧化酶的电子受体，并可能形成一条用于亚砷酸盐解毒的周质电子传递途径。以亚砷酸盐或连二亚硫酸盐作为电子源，亚砷酸盐氧化酶的完全还原需要3 - 4个还原当量。在20 K以下，氧化态的亚砷酸盐氧化酶呈现出g值为2.03、2.01和2.00的电子顺磁共振（EPR）信号，其积分强度约为0.4个自旋/蛋白。由于用57Fe富集导致该EPR信号变宽，产生此信号的中心必定含有铁。最有可能的候选物是一个[4Fe - 4S]高电位铁蛋白中心或一个[3Fe - 4S]中心。在用亚砷酸盐或连二亚硫酸盐还原蛋白后，氧化态亚砷酸盐氧化酶中观察到的EPR信号消失。与此同时，出现了一个菱形EPR信号（g = 2.03、1.89、1.76），它与Rieske型[2Fe - 2S]簇的信号相似，自旋定量为一个自旋/蛋白。
