Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.

Each flavoprotein subunit (PchF) of p-cresol methylhydroxylase (PCMH) has flavin adenine dinucleotide (FAD) covalently tethered to Tyr384. The PCMH structure suggests that Arg474 in PchF is required for self-catalytic covalent flavinylation and for substrate oxidation. The replacement of Arg474 with Lys was carried out to probe the subtleties of the role of Arg474 in these processes.