The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.
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.
Anderson
G L
GL
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Williams
J
J
Hille
R
R
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013104:Spheroplasts / Q000201:enzymology; D013440:Sulfides / Q000032:analysis; D013816:Thermodynamics
D013440:Sulfides; D008982:Molybdenum; D007501:Iron; D010088:Oxidoreductases; C078179:arsenite oxidase