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Salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.1) in Pseudomonas putida catalyzed hydroxylation of the substrate analogue, salicylaldehyde, to form catechol and formate with stoichiometric consumption of NADH and O2. Consequently, a study of primary product derived from the carboxyl group of the authentic substrate, salicylate, was undertaken. The experimental results revealed that CO2 not H2CO3, was produced first.

1. 1. Salicylate hydroxylase (salicylate, NADH:oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) from Pseudomonas putida forms an enzyme-substrate complex with salicylate. 2. 2. The complex could be detected by a new absorption maximum around 480 nm. By spectrophotometric titration, it was found that a molar ratio of apoenzyme, FAD and salicylate in the complex was I:I:I. 3. 3. The complex was more stable than the holoenzyme under any tested conditions, i.e., heat, acid and proteinase treatments. 4. 4. The FAD moiety of the complex was reduced with NADH under anaerobic conditions, and the reoxidation of the reduced complex with air resulted in product formation. The stoichiometric relation in each reaction was demonstrated by using substrate level amounts of the enzyme. A mechanism for salicylate hydroxylation reaction is proposed.

1. 1.|Binary and ternary complexes between apoenzyme of salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) and components such as FAD, substrate and NADH were fluorometrically studied to determine dissociation constant and stoichiometric ratio of each component. This was based on the changes in fluorescence intensity when the apoenzyme was mixed with these components. 2. 2.|A protein-denaturing agent or an alkali treatment was shown to depress the fluorescence emission of the apoenzyme with the red shift of the maximum. 3. 3.|The addition of FAD to the apoenzyme resulted in a marked decrease in both FAD and protein fluorescences. 4. 4.|Either substrate or NADH formed 1 :1 complex with the apoenzyme. This was indicated by both quenching of protein fluorescence and enhancement of substrate or NADH fluorescence. 5. 5.|The holoenzyme combined specifically with substrate to form a fluorescent ternary complex in which the ratio of apoenzyme, FAD and substrate was 1:1:1. 6. 6.|Upon formation of these complexes, the blue shift occurred in the fluorescence spectrum of protein, salicylate, NADH or FAD.

The reaction mechanism of salicylate hydroxylase (salicylate, NADH:oxygen oxidoreductase(1-hydroxylating, 1-decarboxylating)) from Pseudomonas putida has been analyzed kinetically by flow rapid reaction techniques. The rate of binding of salicylate to the enzyme in the oxidized state is much faster than that to the enzyme in the reduced state. The kinetic behavior of the flavin moiety of salicylate hydroxylase is influenced by the presence of the substrate to be hydroxylated. In the presence of salicylate, both the NADH reduction of the enzyme and the reoxidation of the reduced enzyme with molecular oxygen are most simply interpreted as being two-step processes, in which one step obeys first-order kinetics. On the other hand, the same reactions in the absence of salicylate obey second-order kinetics and are most simply interpreted as one-step processes. From the comparison with the kinetic parameters in the presence and absence of salicylate, it is suggested that the enzyme-salicylate complex is a much more efficient system than the free enzyme for accepting electrons from NADH. A substrate analogue, benzoate, also facilitates the reduction of the enzyme-bound flavin by NADH. Based on these kinetic analyses, a mechanism of the salicylate hydroxylase reaction is discussed.