Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

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.