THE CYTOCHROME SUBUNIT IS NECESSARY FOR COVALENT FAD ATTACHMENT TO THE FLAVOPROTEIN SUBUNIT OF P-CRESOL METHYLHYDROXYLASE

When p-cresol methylhydroxylase (PCMH) is expressed in its natural hos t Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escheri chia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr(384) of the alpha subunit and the correct alpha(2) beta(2) form o f the enzyme is assembled. The apoflavoprotein has been expressed in E . coli in the absence of the beta cytochrome c subunit and purified, W hile noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs. Covalent flavinylation requ ires one molecule each of FAD and cytochrome for each flavoprotein sub unit. The flavinylation process leads to the 2-electron-reduced form o f covalently bound FAD, and the resulting alpha(2) beta(2) enzyme is i dentical to wild-type PCMH. This work presents clear evidence that cov alent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation o f FAD or of the protein, This work is the first to define the basic ch emistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mech anism of this process, It also demonstrates, for the first time, the a bsolute requirement for a partner subunit in the post-translational mo dification of a protein, It is proposed that the covalent FAD bond to Tyr(384) and the phenolic portion of this Tyr are part of the essentia l electron transfer path from FAD to heme.