Properties of Intermediates in the Catalytic Cycle of Oxalate Oxidoreductase and Its Suicide Inactivation by Pyruvate

Abstract

Oxalate:ferredoxin oxidoreductase (OOR) is an unusual member of the thiamine pyrophosphate (TPP)-dependent 2-oxoacid:ferredoxin oxidoreductase (OFOR) family in that it catalyzes the coenzyme A (CoA)-independent conversion of oxalate into 2 equivalents of carbon dioxide. This reaction is surprising because binding of CoA to the acyl-TPP intermediate of other OFORs results in formation of a CoA ester, and in the case of pyruvate:ferredoxin oxidoreductase (PFOR), CoA binding generates the central metabolic intermediate acetyl-CoA and promotes a 105-fold acceleration of the rate of electron transfer. Here we describe kinetic, spectroscopic, and computational results to show that CoA has no effect on catalysis by OOR and describe the chemical rationale for why this cofactor is unnecessary in this enzymatic transformation. Our results demonstrate that, like PFOR, OOR binds pyruvate and catalyzes decarboxylation to form the same hydroxyethylidine–TPP (HE–TPP) intermediate and one-electron transfer to generate the HE–TPP radical. However, in OOR, this intermediate remains stranded at the active site as a covalent inhibitor. These and other results indicate that, like other OFOR family members, OOR generates an oxalate-derived adduct with TPP (oxalyl-TPP) that undergoes decarboxylation and one-electron transfer to form a radical intermediate remaining bound to TPP (dihydroxymethylidene–TPP). However, unlike in PFOR, where CoA binding drives formation of the product, in OOR, proton transfer and a conformational change in the “switch loop” alter the redox potential of the radical intermediate sufficiently to promote the transfer of an electron into the iron–sulfur cluster network, leading directly to a second decarboxylation and completing the catalytic cycle.

A Reverse TCA Cycle 2-Oxoacid:Ferredoxin Oxidoreductase that Makes C-C Bonds from CO2

“…Divided by substrate specificity, OFORs can be categorized into 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), 9 pyruvate:ferredoxin oxidoreductase (PFOR), 10 2-oxoisovalerate:ferredoxin oxidoreductase (VOR), 11 indopyruvate:ferredoxin oxidoreductase (IOR), 12,13 and oxalate oxidoreductase (OOR) 6,14 (Figure S2). Notably, OFORs adapt different oligomeric states ( Figure 1C) and are mostly coenzyme A (CoA)-dependent except for OOR, 15 which is specific for cleaving oxalate into CO 2 without CoA.…”

“…Notably, OFORs adapt different oligomeric states ( Figure 1C) and are mostly coenzyme A (CoA)-dependent except for OOR, 15 which is specific for cleaving oxalate into CO 2 without CoA. 6,14 Although PFOR and OGOR from the rTCA cycle engage in CO 2 fixation, on many occasions, OFORs catalyze the reversal reaction to oxidize a 2-oxoacid and provide low-potential electrons for downstream reactions, including sulfate reduction, 16 dinitrogen reduction, 17 and aromatic compound reduction. 18 Yet the basis for why OFORs display preferences for either CO 2 reduction or 2-oxoacid oxidation in any specific pathway is still unknown.…”

“…7,8 Electronic repulsion between the thiolate of CoA and the radical intermediate shifts the equilibrium toward electron transfer and product formation. 6,7 Acetyl-CoA is released in the last step, and electrons housed on the internal [4Fe-4S] clusters are delivered to an external Fd (or Fds), which are then used in other pathways. (C) The domain arrangements of structurally characterized OFORs.…”

“…(D) The proposed reaction mechanism of carbon fixation and 2-oxoglutarate oxidation by OGOR based on studies from MtPFOR. [6][7][8] Steps involving Fdbased electron transfer are labeled with teal, and those involving internal electron transfer are labeled with red.…”

Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria

“…4). These were based on previous reports of ThDP-dependent enzymes that catalyze the oxidative decarboxylation of pyruvate (19 -21) and those that form radical ThDP intermediates (22)(23)(24)(25)(26)(27). Following decarboxylation of LThDP, one-electron transfer from the C2␣-carbanion/ enamine 1 to molecular oxygen results in the formation of a C2-(␣-hydroxy)-ethylidene-ThDP radical cation (ThDPenamine radical) 2 and superoxide.…”