NDLI: Role of Cysteine 337 and Cysteine 340 in Flavoprotein That Functions as NADH Oxidase from Amphibacillus xylanus Studied by Site-directed Mutagenesis

Content Provider	Journal of Biological Chemistry (JBC)
Author	Ohnishi, Kenji Niimura, Youichi Hidaka, Makoto Masaki, Haruhiko Suzuki, Hideo Uozumi, Takeshi Nishino, Takeshi
Abstract	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 FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.
Related Links	http://www.jbc.org/content/270/11/5812.abstract
Ending Page	5817
Starting Page	5812
Page Count	6
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	11
Volume Number	270
DOI	10.1074/jbc.270.11.5812
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1995-03-17
Access Restriction	Open
Subject Keyword	Enzymology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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