Understanding orchestrated chemical reactions in toluene/o-xylene monooxygenase from pseudomonas sporium OX1

Chapter 1. Geometric and Functional Versatility of Carboxylate-Bridged Nonheme- Diiron Motifs: sMMO and ToMO. Several metalloenzymes utilize a carboxylate-bridged non-heme diiron motif for dioxygen activation. Despite their conserved diiron active site structures and mechanisms of dioxygen activation, they catalyze a wide range of chemical transformations. These observations suggest that diiron-containing enzymes have distinct active sites and secondary/tertiary environments that are tuned for their dedicated biological functions. Detailed studies of two diiron-containing enzymes in the family of bacterial multicomponent monooxygenases (BMMs), soluble methane monooxygenase (sMMO) and toluene/o-xylene monooxygenase (ToMO), are described. The functions and structures of the three or four components of sMMO and ToMO are summarized. Distinctly different dioxygen activation chemistry and hydrocarbon specificity is observed for these two enzymes. A comparison of these two enzymes provides insight into the evolution of diironcontaining enzymes as well as their differing chemical mechanisms of catalysis. Chapter 2. Role of an Active Site Threonine in the Determination of Distinctive Dioxygen Reactivity in Toluene/o-Xylene Monooxygenase Hydroxylase. Dioxygen activation of toluene/o-xylene monooxygenase hydroxylase (ToMOH) exhibits the formation of a diiron(III) intermediate having unprecedented spectroscopic properties. To evaluate whether an active site threonine plays a role in the determination of the dioxygen chemistry in ToMOH, a T201S variant was prepared by site-directed mutagenesis. We reported the observation of a novel intermediate in the reaction of reduced ToMOH T201 S variant with dioxygen in the presence of its cognate regulatory protein (ToMOD). This species, T201 peroxo, is the first oxygenated intermediate of any toluene monooxygenase to display an optical band. The optical and M6ssbauer spectroscopic properties of the intermediate allowed us to assign it as a peroxodiiron(III) species, similar to Hperoxo in soluble methane monooxygenase hydroxylase (sMMOH). This result indicates that mutation of the T201 to serine altered the dioxygen chemistry of ToMOH in part to be more similar to that of sMMOH. Computational studies suggest that the T201 mutation can greatly perturb the energetics of the enzyme, which might be responsible for the distinct dioxygen reactivity of sMMOH and ToMOH. Structures of the oxygenated intermediates of ToMOH are proposed. Chapter 3. Role of an Active Site Threonine in the Kinetics of Dioxygen Activation in Toluene/o-Xylene Monooxygenase Hydroxylase. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of toluene/o-xylene monooxygenase hydroxylase (ToMOH), T201S, T201G, T201C, and T201V variants of this enzyme were prepared by site-directed mutagenesis. X-ray crystal structures of all variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was monitored by stopped-flow UV-vis and M6ssbauer spectroscopy. These studies demonstrated that the same dioxygen activation mechanism is preserved in the T201S, T201C, and T201G variants; however, both formation and decay kinetics of a peroxodiiron(III) intermediate, T201peroxo, were greatly altered, revealing that the T201 residue is critically involved in dioxygen activation. Rate-limiting steps in dioxygen activation of the T201S, T201C, and T201G variants were identified, revealing that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. The role of the active site threonine residue in ToMOH is analogous to that of cytochrome P450 monooxygenases, suggesting it as a general threonine-dependent process in Nature to control proton transfer.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2011

Author dc:creator

• Song, Woon Ju

Advisor dc:contributor.advisor

• Stephen J. Lippard.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/65476

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/65476