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

Each flavoprotein subunit (alpha or PchF) of the alpha (2)beta (2) 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 (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-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 bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron 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 approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E-m,E-7 for covalently bound FAD in PCMH is 84 mV, 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 forms of PchF, Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction 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. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad).PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K-m for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E-m,E-7 Of the flavin, Contributions to this effect likely result from conformational changes.