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The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

Open AccessPublished:November 25, 1992DOI:https://doi.org/10.1016/S0021-9258(18)35891-5
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      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.

      References

        • Moore S.A.
        • Moennich D.M.C.
        • Gresser M.J.
        J. Biol. Chem. 1983; 258: 6266-6271
        • Coddington K.
        Toxicol. Environ. Chem. 1986; 11: 281-290
        • Gresser M.J.
        J. Biol. Chem. 1981; 256: 5981-5983
        • Crane R.K.
        • Lipmann F.
        J. Biol. Chem. 1953; 201: 235-243
        • Oehme F.W.
        Toxicology. 1972; 5: 215
        • Knowles F.C.
        • Benson A.A.
        Trends Biochem. Sci. 1983; 8: 178-180
        • Goyer R.A.
        Klaassen C.D. Amdur M.O. Doull J. Casarett and Doull's Toxicology. 3rd Ed. Macmillan, New York1986: 582-635
        • Rosenberg H.
        • Gerdes R.G.
        • Chegwidden K.
        J. Bacteriol. 1977; 131: 505-511
        • Willsky G.R.
        • Malamy M.H.
        J. Bacteriol. 1980; 144: 356-365
        • Willsky G.R.
        • Malamy M.H.
        J. Bacteriol. 1980; 144: 366-374
        • Silver S.
        • Keach D.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6114-6118
        • Mobley H.L.T.
        • Rosen B.P.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6119-6122
        • Silver S.
        • Nucifora G.
        • Chu L.
        • Misra T.K.
        Trends Biochem. Sci. 1989; 14: 76-80
        • Ishinishi N.
        • Tsuchiya K.
        • Vahter M.
        • Fowler B.A.
        Friberg L. Nordberg G.F. Vouk V.B. Handbook of Toxicology of Metals. Elsevier, New York1986: 43-83
        • Benko V.
        • Symon K.
        • Chladek V.
        • Pihrt J.
        Environ. Res. 1977; 13: 386-395
        • Vahter M.
        • Envall J.
        Environ. Res. 1983; 32: 13-24
        • Osborne F.H.
        • Erlich H.L.
        J. Appl. Bacteriol. 1976; 41: 295-305
        • Phillips S.E.
        • Taylor M.K.
        Appl. Environ. Microbiol. 1976; 32: 392-399
        • Turner A.W.
        • Legge J.W.
        Austral. J. Biol. Sci. 1954; 7: 479-495
        • Turner A.W.
        Nature. 1949; 164: 76-77
        • Green H.H.
        Rep. Dir. Vet. Res. S. Afr. 1918; 5–6: 593-599
        • Rinderle S.J.
        • Schrier J.
        • Williams J.W.
        Fed. Proc. 1984; 43: 2060
        • Williams J.W.
        • Rinderle S.J.
        • Schrier J.A.
        • Alvey L.J.
        • Tseng K.
        Fed. Proc. 1986; 45: 1660
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        J. Biol. Chem. 1951; 193: 265-275
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Anderson G.L.
        • Howard J.B.
        Biochemistry. 1984; 23: 2118-2122
        • Ljones T.
        • Burris R.H.
        Biochemistry. 1978; 17: 1866-1872
        • Fish W.W.
        Methods Enzymol. 1988; 158: 357-364
        • Johnson J.L.
        Methods Enzymol. 1988; 158: 371-382
        • Kaback H.L.
        Methods Enzymol. 1971; 22: 99-122
        • Hemmerich P.
        Helv. Chim. Acta. 1964; 47: 464-475
        • Mayhew S.G.
        • Foust G.P.
        • Massey V.
        J. Biol. Chem. 1969; 244: 803-810
        • Komai H.
        • Massey V.
        • Palmer G.
        J. Biol. Chem. 1969; 244: 1692-1700
        • Mitra S.
        • Bersohn R.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6811-6907
        • Ryden L.
        Lontie R. Copper Proteins and Copper Enzymes. I. CRC Press, Boca Raton, FL1984: 157-182
        • Husain M.
        • Davidson V.L.
        J. Biol. Chem. 1985; 260: 14626-14629
        • Chandrasekar R.
        • Klapper M.H.
        J. Biol. Chem. 1986; 261: 3616-3619
        • Silver S.
        • Budd K.
        • Leahy K.M.
        • Shaw W.V.
        • Hammond D.
        • Novick R.P.
        • Willsky G.R.
        • Malamy M.H.
        • Rosenberg H.
        J. Bacteriol. 1981; 146: 983-996
        • Johnson J.L.
        • Hainline B.E.
        • Rajagopalan K.V.
        J. Biol. Chem. 1980; 255: 1783-1786
        • Gutteridge S.
        • Tanner S.J.
        • Bray R.C.
        Biochem. J. 1978; 175: 887-897
        • Hille R.
        • Massey V.
        Spiro T.G. Molybdenum Enzymes. Wiley-Interscience, New York1985: 443-518
        • Fee J.A.
        • Findling K.L.
        • Yoshida T.
        • Hille R.
        • Tarr G.E.
        • Hearshen D. 0.
        • Dunhamn W.R.
        • Day E.P.
        • Kent T.A.
        • Munck E.
        J. Biol. Chem. 1984; 259: 124-133
        • Siedow J.N.
        • Power S.
        • de la Roas F.F.
        • Palmer G.
        J. Biol. Chem. 1978; 253: 2392-2399
        • Malkin R.
        • Bearden A.J.
        Biochim. Biophys. Acta. 1978; 505: 147-181
        • Ballou D.
        • Batie C.
        King T.E. Mason H.S. Morrison M. Oxidases and Related Redox Systems. Alan R. Liss, New York1988: 211-226
        • Sauber K.
        • Frohner C.
        • Rosenberg G.
        • Eberspcher J.
        • Lingens F.
        Eur. J. Biochem. 1977; 74: 89-97
        • Twilfer H.
        • Bernhardt F.H.
        • Gersond K.
        Eur. J. Biochem. 1981; 119: 595-601
        • Geary P.J.
        • Saboowalla F.
        • Patil D.
        • Cammack D.
        Biochem. J. 1984; 217: 667-673
        • Rieske J.S.
        • MacLennan D.H.
        • Coleman R.
        Biochem. Biophys. Res. Commun. 1964; 15: 338-344
        • Fox B.G.
        • Surerus K.K.
        • Munck E.
        • Lipsomb J.D.
        J. Biol. Chem. 1988; 263: 10553-10556
        • Davis J.C.
        • Averill B.A.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4623-4627
        • Antanaitis B.C.
        • Aisen P.
        • Lilienthal H.R.
        J. Biol. Chem. 1983; 258: 3166-3172
        • Cramer S.P.
        • Hille R.
        J. Am. Chem. Soc. 1985; 107: 8164-8169
        • Porras A.G.
        • Palmer G.
        J. Biol. Chem. 1982; 257: 11617-11626
        • Turner A.W.
        Austral. J. Biol. Sci. 1954; 7: 452-478
        • Turner A.W.
        • Legge J.W.
        Austral. J. Biol. Sci. 1954; 7: 479-495
        • Legge J.W.
        • Turner A.W.
        Austral. J. Biol. Sci. 1954; 7: 496-503
        • Legge J.W.
        Austral. J. Biol Sci. 1954; 7: 504-514
        • Trumpower B.L.
        Biochim. Biophys. Acta. 1981; 639: 129-155
        • Palmer G.
        Boyer P.D. The Enzymes. 12. Academic Press, Orlando, FL1975: 1-56
        • Bard A.J.
        • Parsons R.
        • Jordon J.
        Standard Potentials of Aqueous Solutions. Marcel Dekker, New York1985
        • Lappin A.G.
        • Segal M.G.
        • Weatherburn D.C.
        • Henderson R.A.
        • Sykes A.G.
        J. Am. Chem. Soc. 1979; 101: 2302-2306