SUBUNIT INTERACTIONS CHANGE THE HEME ACTIVE-SITE GEOMETRY IN PARA-CRESOL METHYLHYDROXYLASE

The enzyme p-cresol methylhydroxylase [4-cresol:(acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1] contains two subunits: a cytochrome c (electron transfer) subunit (cytochrome c(pc)) and a flavin (catalytic) subunit. When these subunits are separated by isoelectric focusing, a stable cytochrome subunit is obtained. Significant differences are observed between the one-dimensional NMR spectra of oxidized cytochrome c(pc) and of oxidized p-cresol methylhydroxylase. Analysis of the two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY) spectrum of reduced cytochrome c(pc) suggests that the axial ligand, Met-50, of the stable subunit reorients by a rotation about the C-gamma-S-delta-bond when cytochrome c(pc) binds to the flavin subunit. This reorientation must result in a change in bonding at the heme, which is reflected both in the paramagnetically shifted resonances and in the redox potential. p-Cresol methylhydroxylase thereby provides an interesting example of the coupling of subunit interactions to active-site structure and reactivity.