Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis.
The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.
D000596:Amino Acids / Q000032:analysis; D004227:Dithionite / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013054:Spectrophotometry, Atomic
D000596:Amino Acids; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D010544:Peroxidases; C021845:NAD+ peroxidase