Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase.
Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.
D000332:Aerobiosis; D000693:Anaerobiosis; D003352:Corynebacterium / Q000201:enzymology*; D004227:Dithionite / Q000494:pharmacology; D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011485:Protein Binding; D050879:Sarcosine Oxidase; D013053:Spectrophotometry; D013447:Sulfites / Q000494:pharmacology
D005415:Flavins; D013447:Sulfites; D004227:Dithionite; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase