Structural insight into protein complex formation and reaction mechanism of toluene 4-monooxygenase

This thesis reports the structural advances on the diiron hydroxylase toluene 4-monooxygenase (T4MO), a multicomponent enzyme complex that catalyzes the regiospecific oxidation of toluene to form p-cresol. T4MO includes an ∼ 200 kDa hydroxylase with an (alphabetagamma) 2 configuration which houses a diiron center for catalysis, T4moC, a Rieske-type ferredoxin, T4moF, an oxidoreductase and T4moD, a cofactorless effector protein.;Here, the first stoichiometric hydroxylase-effector protein structure is reported providing insight into a number of phenomena associated with this protein complex. Coordinated changes occur in and extending ∼ 25 A from the active site on the helices that house the diiron center to create an active site pocket poised for catalysis.;Additionally, the structure of the T4moH-T4moD complex revealed significant rearrangements of a number of conserved residues, including Thr-201, Asn-202 and Gln-228. Through a series of catalytic and structural studies, the role of each residue was assessed. Thr-201 was found to stabilize a peroxo moiety during T4moH steady state turnover and slow product formation in pre-steady state reactions. Mutation of Asn-202 to alanine resulted in ∼ 10-fold lower steady-state activity which could be attributed to an impaired ability of T4moC to reduce the diiron center. Mutation of Gln-228 resulted in lowered steady-state rates likely stemming from an altered water network within the active site.;Lastly, a peroxide shunt reaction was observed in the T4MO system with an ∼ 600-fold lower steady state activity. A structure of hydrogen peroxide soaked crystals of the T4moH-T4moD complex revealed a diatomic ligand at the active site, providing insight into the peroxide shunt reaction and canonical pathway.