Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase.

Each flavoprotein subunit (α or PchF) of the α2β2 flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (β or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchFC). Dramatic increases in the two-electron Em,7 (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron Em,7 value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchFC. The Em,7 values increased by approximately 20 and 45 mV, respectively, when PchFC and PchF[Y384F]FAD associated with PchC. The two-electron Em,7 for covalently bound FAD in PCMH is 84 mV, the h...