Citation

Streit BR, Mattice JR, Prussia GA, Peters JW, DuBois JL. J. Biol. Chem. 2019; 294(13): 5137-5145.

Copyright

(Copyright © 2019, American Society for Biochemistry and Molecular Biology)

DOI

10.1074/jbc.RA118.005554

PMID

30696768

PMCID

PMC6442062

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.

Language: en

Keywords

Kinetics; Oxidation-Reduction; Substrate Specificity; microbiology; Catalytic Domain; Models, Molecular; Carbon Dioxide; NADP; 2-KPCC; C–S bond cleavage; carbon fixation; carboxylase; disulfide; disulfide oxidoreductase; flavin adenine dinucleotide (FAD); Ketone Oxidoreductases; nucleophilic thiolate; oxidase; Xanthobacter