Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

Knoot, Cory J.; Purpero, Vincent M.; Lipscomb, John D.

doi:10.1073/pnas.1419118112

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

Journal Article · Sun Dec 28 23:00:00 EST 2014 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1419118112· OSTI ID:1170026

Knoot, Cory J. ^[1]; Purpero, Vincent M. ^[1]; Lipscomb, John D. ^[1]

Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Biochemistry Molecular Biology and Biophysics. Center for Metals in Biocatalysis

Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe³⁺ to activate O₂ and catecholic substrates for reaction. The inability of Fe³⁺ to directly bind O₂ presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated in this paper using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe³⁺ species, and the anhydride-Fe³⁺ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe²⁺-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe²⁺ intermediate that could bind O₂ are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Finally, structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Minnesota, Minneapolis, MN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Inst. of Health (NIH) (United States)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1170026

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 2 Vol. 112; ISSN 0027-8424

Publisher:: National Academy of Sciences, Washington, DC (United States)Copyright Statement

Country of Publication:: United States

Language:: ENGLISH

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