Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme

Abstract

An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van''t Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15.degree. C. Above this temperature, KD is characterized by a positive .DELTA.H value of 12.6 kcal mol-1; below 15.degree. C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.