Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OX1

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In the present study we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H2O2) generation. Single turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ~50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to generation of H2O2 in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed. Bacterial multicomponent monooxygenases (BMMs1) are remarkable enzymes that orchestrate a series of electron transfer and substrate activation events in order to prime dioxygen for donation of a single oxygen atom into a C–H bond or across a C=C double bond (1,2). Proteins belonging this family are subdivided into four classes, soluble methane monooxygenases (sMMOs), phenol hydroxylases (PHs), alkene monooxygenases (AMOs), and four-component alkene/arene monooxygenase (TMOs), based on substrate preference and sequence homology (3,4). The ability of BMMs to generate potent oxidizing species without damaging their active sites or consuming electrons in a futile manner depends on the dynamic involvement of three or more protein components: a 200-255 kDa dimeric hydroxylase that houses two copies of a carboxylate-bridged diiron catalytic center; a 38-45 kDa reductase that accepts electrons from NADH and shuttles them though its flavin and [2Fe-2S] cluster cofactors into the hydroxylase diiron sites; and a 10-16 kDa regulatory protein that couples electron consumption to hydrocarbon oxidation (1,2,5). For ToMO, an additional 12 kDa Rieske protein acts as an electron conduit between the reductase and the