A single strain of Pseudomonas cepacia cells was differentially induced to synthesize salicylate hydroxylase, 3-hydroxybenzoate 6-hydroxylase, or 4-hydroxybenzoate 3-hydroxylase. A procedure was developed for the purification of 3-hydroxybenzoate 6-hydroxylase to apparent homogeneity. The purified hydroxylase appears to be a monomer with a molecular weight of about 44,000 and exhibits optimal activity near pH 8. The hydroxylase contains one FAD per enzyme molecule and utilizes NADH and NADPH with similar efficiencies. The reaction stoichiometry for this enzyme has been determined. In comparison with other aromatic flavohydroxylases, this enzyme is unique in inserting a new hydroxyl group to the substrate at a position para to an existing one.

Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes.

A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-[4-2H]NADH and (4R)-[4-3H]NADH as substrates. The DV effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD+ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small (approximately 2-fold) preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.