The flavin prosthetic group (FAD) of the aromatic hydroxylases melilotate hydroxylase (EC 126.96.36.199) and phenol hydroxylase (EC 188.8.131.52) was replaced by 1-deaza-FAD (carmon substituted for nitrogen at position 1). Neither modified enzyme could hydroxylate its substrate, both catalyzed the oxidation of NAD(P)H to NAD(P)+ and H2O2. The rate of the reduction of the enzymes by NAD(P)H was increased by the binding of substrate. Both enzymes formed a detectable flavin C(4a) hydroperoxide intermediate upon reaction of the reduced enzyme-substrate complex with oxygen. Reduced 1-deaza-FAD phenol hydroxylase also showed a detectable C(4a) hydroperoxide intermediate when reacted with oxygen in the absence of substrate. The C(4a) hydroperoxide of 1-deaza-FAD phenol hydroxylase, in the absence of phenol, decayed to an intermediate which showed a perturbed oxidized enzyme spectrum, E*(ox). This intermediate in turn decayed to give the original oxidized enzyme. In the presence of phenol, a second oxidized species with a perturbed spectrum, intermediate X, was apparent after formation of the flavin C(4a) hydroperoxide and before E*(ox) formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the E*(ox) to E(ox) conversion was not in the catalytic cycle. During turnover E*(ox) was reduced by NADPH.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology