Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or Pch F(2); M-r = 119 kDa) with a cytochrome monomer (beta, PchC; M-r = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalentl y. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC) ), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol ox idation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rap idly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact P CMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 ele ctrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchFC accepted on ly 2 electron equiv/mol. This is explained by extremely slow release of 4-h ydroxybenzyl alcohol from reduced PchFC. 4-Hydroxybenzyl alcohol rapidly re duced PchFC, producing 4-hydroxybenzaldehyde. It was demonstrated that p-cr esol has a charge-transfer interaction with FAD when bound to oxidized PchF (NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyd e have charge-transfer interactions with FAD when bound to either PchFC or PchF(NC). This is the first example of a "wild-type" flavoprotein, which no rmally has covalently bound Ravin, to bind flavin noncovalently in a stable , redox-active manner.