Influence of Anions on the Reaction Mechanism of the Two‐Component Flavoenzyme Styrene Monooxygenase

Styrene monooxygenase component A (SMOA) is a FAD‐dependent styrene epoxidase, which catalyzes the first step in styrene catabolism by Pseudomonas putida S12. The reduced FAD required for the oxidative half reaction of SMO is supplied by styrene monoxygenase component B (SMOB), an NADH requiring flavin reductase. We have cloned and expressed native SMOA, and report here a two‐step method for the purification of native, recombinant enzyme by using ion exchange and dye‐ligand affinity chromatography. The native enzyme shows very similar kinetic behavior to the N‐terminally histidine‐tagged version of SMOA previously reported by our laboratory. Monovalent anions have been previously shown to have a significant role in stabilizing flavin‐peroxide intermediates in the reaction mechanism of the single‐component flavin aromatic ring hydroxylases. Results from our studies of the role of anions in the reduction and oxygenation reactions of SMO and in the efficiency of reduced flavin‐transfer from SMOB to SMOA will be presented.