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 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.
Engst
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Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Kuusk
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Efimov
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Cronin
C N
CN
McIntire
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D001426:Bacterial Proteins; D001547:Benzaldehydes / Q000737:chemistry; D001665:Binding Sites; D003580:Cytochromes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004227:Dithionite / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005420:Flavoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D010453:Peptide Synthases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D017186:Titrimetry
D001426:Bacterial Proteins; D001547:Benzaldehydes; D003580:Cytochromes; D005420:Flavoproteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D006899:Mixed Function Oxygenases; C049502:4-cresol dehydrogenase (hydroxylating); D010453:Peptide Synthases; C401840:aryl-4,2-bisthiazoline synthetase, Pseudomonas aeruginosa; C011483:4-hydroxybenzaldehyde