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 this 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 H2O2 formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334)CEINGE - Biotecnologie Avanzate (Italy