Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: Gated substrate entry and proton relays support the proposed catalytic mechanism

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enz yme first catalyzes the oxidation of p-eresol to p-hydroxybenzyl alcohol, u tilizing one atom of oxygen derived from water, and yielding one molecule o f reduced FAD. The reducing electron equivalents are then passed one at a t ime from the flavin cofactor to the heme cofactor by intramolecular electro n transfer, and subsequently to cytochrome oxidase within the periplasmic m embrane via one or more soluble electron carrier proteins. The product, p-h ydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzal dehyde. The fully refined X-ray crystal structure of PCMH in the native sta te has been obtained at 2.5 Angstrom resolution on the basis of the gene se quence. The structure of the enzyme-substrate complex has also been refined , at 2.75 Angstrom resolution, and reveals significant conformational chang es in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route f or substrate access to the site has been identified and is shown to be gove rned by a swinging-gate mechanism. Two possible proton transfer pathways, t l;at may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydro gen bonding interactions between the flavoprotein and cytochrome subunits t hat stabilize the intramolecular complex and may contribute to the electron transfer process have been identified. (C) 2000 Academic Press.