Effects of noncovalent and covalent FAD binding on the redox and catalyticproperties of p-cresol methylhydroxylase

Each flavoprotein subunit (alpha or PchF) of the alpha (2)beta (2) flavocyt ochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzy l alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric n oncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subun it (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with w ild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E-m,E-7 (NHE) values for FAD were observed when it bou nd noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electro n E-m,E-7 value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e,, PchF(C). The E-m,E-7 values increased by approximate ly 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated w ith PchC. The two-electron E-m,E-7 for covalently bound FAD in PCMH is 84 m V, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E-m,E-7, NHE) for FAD were measured also for various for ms of PchF, Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrat es was similar to that observed previously for PchF containing noncovalentl y bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduc tion of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad). PchC, the mutant enzyme containing the flavin radical and reduced heme. The se experiments also revealed a slow reduction of unassociated PchC(ox) by P chF[Y384F]FAD(rad).PchC. Steady-state kinetic studies of the reaction of PC MH[Y384F] with p-cresol indicated that the K-m for this substrate was uncha nged relative to that of PCMH, but that the k(cat) was diminished by an ord er of magnitude. The data indicate that the covalent attachment of FAD to P chF assists catalysis by raising the E-m,E-7 Of the flavin, Contributions t o this effect likely result from conformational changes.