Abstract
2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.
Highlights
NADPH:2-ketopropyl– coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other disulfide– containing oxidoreductases (DSORs) by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site
The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine
NADPH:2-ketopropyl– coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial enzyme that catalyzes the direct fixation of CO2 into biomass as part of a pathway for metabolizing small alkenes, including propylene gas [3]
Summary
The reductive half-reaction in 2-KPCC and all DSORs starts with NADPH binding and ends with the generation of the two-electron–reduced active site (Fig. 1) [4, 10] This is the catalytically competent form of the enzyme that binds to and reduces substrate. The data exhibited complete independence of the rate of this step on the heavy isotope This experiment showed conclusively that no protons exchange places in the reduced 2-KPCC active site as CysINT cross-links with BES. In light of the predicted, more nucleophilic reduced active site of 2-KPCC, relative to typical DSORs, we wondered whether the oxidative half-reaction with 2-KPC (Fig. 4) might proceed rapidly. In contrast to typical DSORs, the oxidative half-reaction limits the overall rate of catalysis for 2-KPCC
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