| Abstract
| - The α2β2 flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida iscomposed of a flavoprotein homodimer (α2 or PchF2; Mr = 119 kDa) with a cytochrome monomer (β,PchC; Mr = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformedwith 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 noncovalently.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 sufficientlyhigh to prevent the determination of the dissociation constant. It was found that p-cresol was virtuallyincapable of reducing PchF with noncovalently bound FAD (PchFNC), whereas 4-hydroxybenzyl alcohol,the intermediate product of p-cresol oxidation by PCMH, reduced PchFNC fairly quickly. In contrast,p-cresol rapidly reduced PchF with covalently bound FAD (PchFC), but, unlike intact PCMH, whichconsumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from4-hydroxybenzyl alcohol), PchFC accepted only 2 electron equiv/mol. This is explained by extremelyslow release of 4-hydroxybenzyl alcohol from reduced PchFC. 4-Hydroxybenzyl alcohol rapidly reducedPchFC, producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interactionwith FAD when bound to oxidized PchFNC, whereas 4-bromophenol (a substrate analogue) and4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchFC or PchFNC.This is the first example of a “wild-type” flavoprotein, which normally has covalently bound flavin, tobind flavin noncovalently in a stable, redox-active manner.
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