A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The K for NADH and oxygen and the k value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Niimura, Y. Nishiyama, D. Saito, H. Tsuji, M. Hidaka, T. Miyaji, T. Watanabe, and V. Massey A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in Amphibacillus xylanus J. Bacteriol., September 15, 2000; 182(18): 5046 - 5051. [Abstract] [Full Text]

				P. Arcari, L. Masullo, M. Masullo, F. Catanzano, and V. Bocchini A NAD(P)H Oxidase Isolated from the Archaeon Sulfolobus solfataricus Is Not Homologous with Another NADH Oxidase Present in the Same Microorganism. BIOCHEMICAL CHARACTERIZATION OF THE ENZYME AND CLONING OF THE ENCODING GENE J. Biol. Chem., January 14, 2000; 275(2): 895 - 900. [Abstract] [Full Text] [PDF]

				Y. Niimura and V. Massey Reaction Mechanism of Amphibacillus xylanus NADH Oxidase/Alkyl Hydroperoxide Reductase Flavoprotein J. Biol. Chem., November 29, 1996; 271(48): 30459 - 30464. [Abstract] [Full Text] [PDF]

				Y. Niimura, L. B. Poole, and V. Massey Amphibacillus xylanus NADH Oxidase and Salmonella typhimurium Alkyl-hydroperoxide Reductase Flavoprotein Components Show Extremely High Scavenging Activity for Both Alkyl Hydroperoxide and Hydrogen Peroxide in the Presence of S. typhimurium Alkyl-hydroperoxide Reductase 22-kDa Protein Component J. Biol. Chem., October 27, 1995; 270(43): 25645 - 25650. [Abstract] [Full Text] [PDF]