Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase
- 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.
- Research Organization:
- Univ. of Minnesota, Minneapolis, MN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Inst. of Health (NIH) (United States)
- Grant/Contract Number:
- AC02-06CH11357; GM24689; GM08700
- OSTI ID:
- 1170026
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 2; ISSN 0027-8424
- Publisher:
- National Academy of Sciences, Washington, DC (United States)Copyright Statement
- Country of Publication:
- United States
- Language:
- ENGLISH
Web of Science
Highly Selective and Catalytic Oxygenations of C−H and C=C Bonds by a Mononuclear Nonheme High‐Spin Iron(III)‐Alkylperoxo Species
|
journal | July 2019 |
Highly Selective and Catalytic Oxygenations of C−H and C=C Bonds by a Mononuclear Nonheme High-Spin Iron(III)-Alkylperoxo Species
|
journal | August 2019 |
Hydrogen atom abstraction by synthetic heme ferric superoxide and hydroperoxide species
|
journal | January 2019 |
Mechanistic insights into ring cleavage of hydroquinone by PnpCD from quantum mechanical/molecular mechanical calculations
|
journal | January 2019 |
Similar Records
Nuclear Resonance Vibrational Spectroscopy Definition of O2 Intermediates in an Extradiol Dioxygenase: Correlation to Crystallography and Reactivity
Intermediate in the O−O Bond Cleavage Reaction of an Extradiol Dioxygenase