Mechanism of reduction of Corynebacterium sarcosine oxidase by dithiothreitol.
The mechanism of the reduction of Corynebacterium sarcosine oxidase [EC 1.5.3.1] by dithiothreitol (DTT) was investigated. The reduction followed biphasic kinetics with second-order rate constants of 54 M-1 X S-1 and 5.4 M-1 X S-1 for the respective phases. When the oxidized enzyme was titrated with sarcosine under anaerobic conditions, no intermediate, such as a semiquinone or a charge-transfer complex, appeared during the reduction of the enzyme. On the other hand, on DTT titration, an intermediate with a semiquinoid character appeared, and its formation was maximum when half of the total FAD was reduced. An oxidized semiapoenzyme, which had lost 45% of the noncovalently-bound FAD present in the native enzyme, also showed biphasic kinetics in the reduction with DTT. The second-order rate constant was found to be 38 M-1 X S-1 for the fast phase. An intermediate was also formed and its concentration, estimated by electron spin resonance (ESR) measurement, was found to agree with that of the noncovalently-bound FAD. In addition, the oxidized semiapoenzyme, which had lost 95% of the noncovalently-bound FAD present in the native enzyme, was reduced with DTT much more slowly than the native enzyme. In this case, the second-order rate constant was found to be 0.4 M-1 X S-1, and no intermediate was observed during the titration with DTT. On the basis of these data, it is suggested that the noncovalently-bound FAD accepts electrons directly from DTT in the fast phase through the semiquinoid form, while the covalently-bound FAD accepts electrons from the reduced noncovalently-bound FAD in the slow phase without forming an intermediate.
D000693:Anaerobiosis; D003352:Corynebacterium / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011485:Protein Binding; D050879:Sarcosine Oxidase; D013053:Spectrophotometry
D005182:Flavin-Adenine Dinucleotide; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D004229:Dithiothreitol