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.