Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.Bacterial multicomponent monooxygenases (BMMs) are a large and heterogeneous family of nonheme di-iron enzymes which share the very interesting ability to activate dioxygen and transfer a single oxygen atom to a wide variety of substrates (13, 23). Aliphatic and aromatic hydrocarbons are converted, respectively, into alcohols and phenols (3,6,15,21,22,38), alkenes are converted into epoxides (8), and sulfur-containing compounds are oxidized into sulfoxides and sulfones (10).As BMMs allow bacteria to grow on hydrocarbons or xenobiotics as the sole source of carbon and energy, several members of this protein family, including the soluble methane monooxygenases (MMOs) (15, 21), alkene monooxygenases (8), phenol hydroxylases (PHs)/toluene 2-monooxygenases (T2MOs) (3,22), and toluene monooxygenases (TMOs) such as toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1 (38) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. strain OX1 (6), have been characterized thoroughly.All these enzymes possess defined substrate specificity, regioselectivity, and enantioselectivity properties. For example, TMOs and PHs perform two consecutive hydroxylation reactions with aromatic rings, but usually TMOs are more efficient in the first hydroxylation step, whereas PHs are more efficient in the second (3, 5). Moreover, each TMO and PH shows its own characteristic regioselectivity. T4MO produces more than 96% p-cresol from toluene (25), whereas ToMO produces a mixture of the three isomers of cresol (5). PHs usually produce a large excess of o-cresol-70 a...