Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.
The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.
Ahmed
S A
SA
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016.
Claiborne
A
A
D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D005415:Flavins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D013431:Sulfates / Q000737:chemistry
D005415:Flavins; D009097:Multienzyme Complexes; D013431:Sulfates; D010544:Peroxidases; C021845:NAD+ peroxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C004850:sodium dithionate