Catalytic properties of streptococcal NADH oxidase containing artificial flavins.
The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.
Ahmed
S A
SA
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016.
Claiborne
A
A
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D004227:Dithionite / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism*; D005415:Flavins / Q000032:analysis*; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry
D001051:Apoenzymes; D001386:Azides; D005415:Flavins; D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D004492:Edetic Acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases