Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p- xylene

Abstract

The present studies address the mechanism of aromatic hydroxylation used by the natural and G103L isoforms of the diiron enzyme toluene 4-monooxygenase. These isoforms have comparable catalytic parameters but distinct regiospecificities for toluene hydroxylation. Hydroxylation of ring-deuterated pxylene by the natural isoform revealed a substantial inverse isotope effect of 0.735, indicating a change in hybridization from sp 2 to sp 3 for hydroxylation at a carbon atom bearing the deuteron. During the hydroxylation of 4-2 H1-and 3,5-2 H2-toluene, similar magnitudes of intramolecular isotope effects and patterns of deuterium retention were observed from both isoforms studied, indicating that the active-site mutation affected substrate orientation but did not influence the mechanism of hydroxylation. The results with deuterated toluenes show inverse intramolecular isotope effects for hydroxylation at the position of deuteration, normal secondary isotope effects for hydroxylation adjacent to the position of deuteration, near-quantitative deuterium retention in m-cresol obtained from 4-2 H1-toluene, and partial loss of deuterium from all phenolic products obtained from 3,5-2 H2-toluene. This combination of results suggests that an active site-directed opening of position-specific transient epoxide intermediates may contribute to the chemical mechanism and the high degree of regiospecificity observed for aromatic hydroxylation in this evolutionarily specialized diiron enzyme.

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

“…On the other hand, formation and deprotonation of the arenium ion intermediate might thus be the major pathway for other substrates such as toluene in T4MO/ToMO, where the occurrence of a crucial arenium cation intermediate was indeed suggested on the basis of docking calculations; 108 although epoxide ring opening mechanism is supported by isotope effect studies. 109 It is interesting to note that the phenylacetyl coenzyme A epoxidase enzyme (Paa), 44 closely related to Box, produces a 1,2-epoxide with no evidence for phenol formation. Our preliminary studies using HCOOOH as the model oxidant indicate that the O LP → π* donation can be also quite significant for the PhCH2C(=O)SMe model substrate as well if the initial electrophilic attack occurs in the C1 (ipso) position, but it seems weak for the C2 (ortho) attack.…”

Biological inorganic chemistry at the beginning of the 21st century

“…One of the highlights in this issue is Larry Que's report of an authentic nonheme ferryl complex, a long-sought species thought by many to play a key role in oxygenation catalytic cycles (69); and other important contributions to ongoing discussions of nonheme iron structures and reactivities are in the Perspectives (60,62,63) as well as in research papers from the laboratories of Marcetta Darensbourg, Brian Fox, Bob Hausinger, Mike Johnson (NO again! ), Julie Kovacs, Don Kurtz, Mike Marletta, and Guenther Winkelmann (70)(71)(72)(73)(74)(75)(76)(77).…”

Structural consequences of effector protein complex formation in a diiron hydroxylase

“…This enzyme hydroxylates toluene with high regiospecificity in the presence of its effector protein, T4moD (3,12). Here, we report X-ray structures of resting T4moH, the stoichiometric complex of resting T4moH with T4moD, and the sodium dithionite-reduced complex to resolutions of 1.9, 1.9, and 1.7 Å respectively [see supporting information (SI) Table S1 for refinement statistics].…”