Purification and characterisation of the NADH: acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWWO of Pseudomonas putida mt‐2

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Abstract
The xylene monooxygenase system encoded by the TOL plasmid pWWO of Pseudomonas putida catalyses the hydroxylation of a methyl side‐chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron‐transfer protein and a terminal hydroxylase, respectively. In this study, the electron‐transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH: cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non‐haem iron and 2 mol/mol of acid‐labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe‐2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH: acceptor reductase was capable of reducing either one‐ or two‐electron acceptors, such as horse heart cytochrome c or 2,6‐dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 μM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro‐R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe‐2S] center occurred with a midpoint redox potential of −171 mV; and the reduction of FAD to FAD˙ (semiquinone form), with a calculated midpoint redox potential of −244 mV. The reduction of FAD˙ to FAD˙˙ (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of −297 mV. The [2Fe‐2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe‐2S]‐depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH→FAD→[2Fe‐2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6‐dichloroindophenol at a reduced rate.