Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.
The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D004396:Coloring Agents; D004563:Electrochemistry; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D062385:Hydroxybenzoates; D007203:Indigo Carmine; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009285:Naphthoquinones; D010084:Oxidation-Reduction; D010226:Parabens; D010269:Paraquat; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013057:Spectrum Analysis; D013816:Thermodynamics
D004396:Coloring Agents; D062385:Hydroxybenzoates; D009285:Naphthoquinones; D010226:Parabens; D005182:Flavin-Adenine Dinucleotide; D007203:Indigo Carmine; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; C038193:4-hydroxybenzoic acid; D010269:Paraquat; C005090:lawsone