1,4-Benzoquinone reductase from basidiomycete Phanerochaete chrysosporium: spectral and kinetic analysis.
The reaction mechanism of a 1,4-benzoquinone reductase from the wood-rotting basidiomycete Phanerochaete chrysosporium was investigated. The native, oxidized, FMN-containing enzyme was reduced quantitatively by NADH and the resulting reduced enzyme was reoxidized in the presence of one equivalent of 2,6-di-methoxy-1,4-benzoquinone (DMBQ). The stoichiometry of NADH oxidation versus DMBQ reduction is 1:1. The enzyme catalyzes the reduction of quinones to hydroquinones by a ping-pong steady-state mechanism. However, inhibition is observed at low NADH concentrations. Quinone products derived from the autooxidation of the unstable compounds 1,2,4-trihydroxybenzene and 5-chloro-2,3,4-trihydroxybenzene also appear to be substrates for the quinone reductase. The enzyme reduces the one-electron acceptors ferricyanide and ferricytochrome c (Cc3+) with rates of 58.4 and 0.08%, respectively, compared to DMBQ. The stoichiometry of NADH oxidation versus ferricyanide reduction is 1:2. In the presence of quinones the rates of Cc3+ and ferricyanide reduction are increased, owing to the nonenzymatic reduction of these acceptors by enzyme-generated hydroquinone products. Dicumarol and Cibacron blue are competitive inhibitors with respect to NADH, with Ki values of 2.1 and 0.30 microM, respectively. Reconstitution of the apoprotein with FMN yields a fully active enzyme at an FMN-to-protein ratio of 2:1, suggesting that the flavin content of the enzyme is two molecules of FMN per dimer.
Brock
B J
BJ
Department of Chemistry,Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Gold
M H
MH
D001487:Basidiomycota / Q000201:enzymology*; D016227:Benzoquinones / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005292:Ferricyanides / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000032:analysis; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014227:Triazines / Q000494:pharmacology
D016227:Benzoquinones; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D005292:Ferricyanides; D006873:Hydroquinones; D011809:Quinones; D014227:Triazines; D009243:NAD; C007931:hexacyanoferrate III; C030511:2,6-dimethoxy-1,4-benzoquinone; C012842:Cibacron Blue F 3GA; D005486:Flavin Mononucleotide; D001728:Dicumarol; C100348:1,4-benzoquinone reductase; D011808:Quinone Reductases