Formation of Hexacoordinate Mn(III) in Bacillus subtilis Oxalate Decarboxylase Requires Catalytic Turnover

Abstract

Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).

Oxygen activation by mononuclear Mn, Co, and Ni centers in biology and synthetic complexes

“…119 Oxalate coordination serves to lower the Mn 3+/2+ redox potential, thus facilitating formation of a Mn/O2 adduct with η 1 -superoxomanganese(III) character. 120 In the OxOx/OxDC mechanisms favored by both Richards and Bornemann, O2 binding is followed by a key proton-coupled electron transfer (PCET) step involving deprotonation of the oxalate ligand and transfer of an electron from oxalate to the nascent Mn(III) center (Scheme 3). 114,121,122 The end-result is a putative superoxomanganese(II)-(oxalate radical anion) intermediate common to both OxOx and OxDC.…”

Biological functions controlled by manganese redox changes in mononuclear Mn-dependent enzymes

“…[49] Heavy atom isotope effect measurements suggest that C-C bond breaking proceeds in a heterolytic fashion [50], presumably from a Mn-bound oxalate radical anion [51]. The latter is almost certainly generated after substrate binding by electron transfer to Mn(III) (Figure 3), and direct observation of Mn(III) during catalytic turnover by high-field, parallel mode EPR measurements support this mechanistic proposal [52]. As in the case of OxOx, however, the molecular species that actually oxidizes the Mn(II) that is present in the purified, recombinant enzyme remains unclear.…”

“…Computational studies support the intermediacy of Mn(III) in the catalytic mechanism [61,62]. Less is known about how the protein modulates the redox properties of the metal, although, as in the case of OxDC [52], the binding of the substrate anion likely stabilizes Mn(III). Unlike the metal centers in MnSOD, OxOx or OxDC (Figures 1-3) in which three conserved histidines are directly coordinated to the metal, a third histidine (His-200) is positioned approximately 3.9 Å away from the Mn center that is thought to hydrogen bond to superoxide during catalysis [63].…”

“…As in the case of OxOx, however, the molecular species that actually oxidizes the Mn(II) that is present in the purified, recombinant enzyme remains unclear. Given that dioxygen is required for catalytic activity, current mechanistic hypotheses postulate metal oxidation by this species to give an initial superoxide radical and Mn(III) complex [50][51][52][53]. Given that the purified Mn(II)-containing enzyme is not oxidized in the absence of oxalate, such a model suggests that binding the negatively charged oxalate monoanion, which is the true substrate [50], modulates the redox properties of the Mn(III)/Mn(II) couple to facilitate dioxygen-mediated oxidation to yield the catalytically active form of the enzyme.…”

Biochemical properties and oxalate‐degrading activity of oxalate decarboxylase from bacillus subtilis at neutral pH

“…Although most of the therapeutic and industrial applications of OxDC depend on its activity under neutral conditions, the enzyme has an optimum pH around 4 and shows a remarkable drop in activity at increasing pH . Thus, efforts should be made to better define the properties of the enzyme at physiological pH, as the basis to improve its oxalate metabolizing activity under physiological conditions.…”