The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers.
The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.
Ahmed
S A
SA
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Claiborne
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine; D004227:Dithionite / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology*; D005420:Flavoproteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry
D005420:Flavoproteins; D009097:Multienzyme Complexes; D004227:Dithionite; D006861:Hydrogen Peroxide; D010544:Peroxidases; C021845:NAD+ peroxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine