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Abstract Oxalate decarboxylase fromBacillus subtilisis a binuclear Mn‐dependent acid stress response enzyme that converts the mono‐anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π‐stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long‐range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5‐hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5‐hydroxytryptophan with its hydroxyl proton removed. 5‐Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N‐terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long‐range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5‐hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.
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Bidentate Substrate Binding Mode in Oxalate Decarboxylase
Oxalate decarboxylase is an Mn- and O2-dependent enzyme in the bicupin superfamily that catalyzes the redox-neutral disproportionation of the oxalate monoanion to form carbon dioxide and formate. Its best-studied isozyme is from Bacillus subtilis where it is stress-induced under low pH conditions. Current mechanistic schemes assume a monodentate binding mode of the substrate to the N-terminal active site Mn ion to make space for a presumed O2 molecule, despite the fact that oxalate generally prefers to bind bidentate to Mn. We report on X-band 13C-electron nuclear double resonance (ENDOR) experiments on 13C-labeled oxalate bound to the active-site Mn(II) in wild-type oxalate decarboxylase at high pH, the catalytically impaired W96F mutant enzyme at low pH, and Mn(II) in aqueous solution. The ENDOR spectra of these samples are practically identical, which shows that the substrate binds bidentate (κO, κO’) to the active site Mn(II) ion. Domain-based local pair natural orbital coupled cluster singles and doubles (DLPNO-CCSD) calculations of the expected 13C hyperfine coupling constants for bidentate bound oxalate predict ENDOR spectra in good agreement with the experiment, supporting bidentate bound substrate. Geometry optimization of a substrate-bound minimal active site model by density functional theory shows two possible substrate coordination geometries, bidentate and monodentate. The bidentate structure is energetically preferred by ~4.7 kcal/mol. Our results revise a long-standing hypothesis regarding substrate binding in the enzyme and suggest that dioxygen does not bind to the active site Mn ion after substrate binds. The results are in agreement with our recent mechanistic hypothesis of substrate activation via a long-range electron transfer process involving the C-terminal Mn ion.
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- Award ID(s):
- 2002950
- PAR ID:
- 10543214
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 29
- Issue:
- 18
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 4414
- Subject(s) / Keyword(s):
- oxalate decarboxylase ENDOR substrate binding bidentate coordination
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules29184414
