The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms.
Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.
Ahmed
S A
SA
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Claiborne
A
A
D004227:Dithionite / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013053:Spectrophotometry
D009097:Multienzyme Complexes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases