flavin-adenine-dinucleotide and 4-hydroxybenzaldehyde

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 +/- 15 kDa and a unique alphaalpha'beta(2) subunit composition, with alpha and alpha' representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and beta representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr(394) of the alpha subunit. In contrast, the alpha' subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K(m) values (1.7 and 2.7 microM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K(ic) (competitive inhibition) value of 18 +/- 9 microM and a K(iu) (uncompetitive inhibition) value of 235 +/- 20 microM. A putative functional model for an unusual PCMH enzyme is presented.

Topics: Bacterial Proteins; Benzaldehydes; Benzyl Alcohols; Catalytic Domain; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cresols; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Flavoproteins; Geobacter; Heme; Mass Spectrometry; Membrane Proteins; Mixed Function Oxygenases; Molecular Weight; Protein Isoforms; Spectrophotometry, Ultraviolet

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(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 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 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 oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Topics: Bacterial Proteins; Benzaldehydes; Binding Sites; Cytochromes; Dithionite; Escherichia coli; Flavin-Adenine Dinucleotide; Flavoproteins; Genetic Vectors; Mixed Function Oxygenases; Peptide Synthases; Recombinant Proteins; Spectrophotometry, Ultraviolet; Substrate Specificity; Titrimetry