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 <i>Bacillus subtilis</i> 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 (κ<i>O</i>, κ<i>O</i>') 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.