Reactions of 1-deaza-FAD-substituted phenol hydroxylase and melilotate hydroxylase

• Published in: The Journal of biological chemistry 1984-02, Vol.259 (3), p.1532-1538
• Main Authors: Detmer, K, Schopfer, L M, Massey, V
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Apoenzymes - metabolism; Biological and medical sciences; Enzymes and enzyme inhibitors; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - pharmacology; Fundamental and applied biological sciences. Psychology; Kinetics; Mixed Function Oxygenases - metabolism; NAD - metabolism; NADP - metabolism; Oxidation-Reduction; Oxidoreductases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43441-7

The flavin prosthetic group (FAD) of the aromatic hydroxylases melilotate hydroxylase (EC 1.14.13.4) and phenol hydroxylase (EC 1.14.13.7) was replaced by 1-deaza-FAD (carbon 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, Eox. 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 Eox formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the Eox to Eox conversion was not in the catalytic cycle. During turnover Eox was reduced by NADPH.