Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/o-Xylene Monooxygenase Hydroxylase

Abstract

Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the 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 explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201 peroxo, were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O 2 activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation and possible structures of oxygenated intermediates are discussed.lippard@mit.edu. SUPPORTING INFORMATION Detailed experimental methods, a table of X-ray data collection, phase determination, and refinement statistics for the structures, a table of product distribution in toluene and o-xylene oxidation, a figure of active sites, a figure of Michaelis-Menten kinetics, time-dependent Mössbauer and UV-vis spectral changes upon the reaction with dioxygen, and the kinetics of T201 peroxo under various conditions in T201 variants. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access NIH-PA Author ManuscriptA role for a strictly conserved threonine residue at the active sites of bacterial multicomponent monooxygenase (BMM) hydroxylases has been discussed for more than a decade. [1][2][3][4][5] In particular, it has been conjectured that the threonine might stabilize a hydrogen-bonding network leading from the protein surface to a metal-bound, active dioxygen moiety, facilitating proton transfer required to generate and stabilize oxygenated intermediates. Such speculation for the function of the threonine residue is based in part by analogy to studies of cytochrome P450 monooxygenase (P450), where a similarly positioned threonine in the distal pocket of the heme site facilitates proton transfer to the distal oxygen atom to form an iron(III) hydroperoxo species, which then converts to an oxoiron(IV) porphyrin π-cation radical. [6][7][8][9] Replacing threonine with alanine uncouples this process, liberating hydrogen peroxide. Unlike P450, however, few studies of diiron monooxygenases have been conducted in sufficient detail to determine whether the threonine residue does play a role during O 2 activation and, if so, whether it operates in a manner similar to that of P450. The present communicatio...

Pathways for Arene Oxidation in Non-Heme Diiron Enzymes: Lessons from Computational Studies on Benzoyl Coenzyme A Epoxidase

“…In several related enzymes, there is a conserved threonine residue near the active site (Thr201 in T4MO/ToMO, Thr213 in MMO), which is postulated to modulate the stability of the oxygenated intermediates 38,74,75 and to play a role as a proton relay. 40 Importantly, this threonine is missing from BoxB; the amino acid in the analogous position is Gly214.…”

Tracking a defined route for O 2 migration in a dioxygen-activating diiron enzyme

“…To assure that the mutations did not greatly perturb the global folding, component interactions, or active site structure of the enzyme system, the iron content and steady-state activity for phenol oxidation were measured. The variants were prepared and characterized as previously described (17). All 13 variants contained 3-4 iron atoms per protein dimer, similar to the content of the wild-type enzyme (SI Appendix, Table S2).…”

“…Heterologous expression and purification of the four ToMO component proteins were carried out as described (18,21). For site-directed mutagenesis, a pET22bðþÞ∕touBEA T201S plasmid was used as the template as described previously (17). Primers used to generate the mutations are listed in SI Appendix, Table S1 and were obtained from Integrated DNA Technologies.…”

“…Instead, we directly measured the dependence of the oxygenation rate of the diiron center in the enzyme on the O 2 concentration under pre-steady-state conditions and compared the results for several variants in which the volume occupied by the side chain of different amino acids along putative O 2 pathways was altered by site-directed mutagenesis. This approach was possible because an oxygenated intermediate (first termed T201S peroxo and later T201 peroxo , λ max ¼ ca: 675 nm) was previously identified in a T201S ToMOH variant during dioxygen activation by reduced ToMOH in the presence of its cognate regulatory protein, ToMOD, and formed at rates that were linearly dependent on the concentration of O 2 (17). This result indicated that O 2 access to the diiron site is a rate-limiting step in T201 peroxo formation under pre-steady-state conditions.…”

“…Two mutations, however, were designed to probe hydrophobicity as well as the size of the amino acids because a polar residue may contribute to the slower rate of dioxygen transfer (25). In the experiments described here, a T201S mutation was introduced to provide a sensor, in the form of an optical transition at 675 nm (17), to detect dioxygen arrival at the diiron sites via formation of the T201 peroxo intermediate. Double or triple variants were prepared and crystallographically characterized to evaluate whether or not the additional mutation would modify the dimensions of various hydrophobic sites along a putative pathway chosen for interrogation.…”