The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

Abstract

Edited by Ruma Banerjee NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO 2 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 CO 2. 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 pK a 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 CO 2 fixation.

The unique Phe–His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S–C bond cleavage

“…Moreover, acetone formation by F501H was comparable in rate to the carboxylation reaction catalyzed by WT 2-KPCC and leading to acetoacetate. These results are consistent with kinetic analyses that showed that Cys–disulfide reduction by NADPH is rate limiting in 2-KPCC ( 3 ) and insensitive to the F501H substitution.…”

“…Our recent work has suggested that the catalytic dyad differences observed in 2-KPCC go beyond that of simply limiting protonation in the promotion of carboxylation. We have shown that 2-KPCC operates through a different key reactive intermediate than GR (5) in which the reduced state of the redox active dithiol exists with a bridged single proton instead of the FAD-CysCT species observed in GR (Scheme 1B, 1C). We can see in our structure of the F501H variant and the structure of GR that the interactions of His with the exchange thiol (CysINT (C82)) would promote the stabilization of the GR-type reduced reactive intermediate relative to the 2-KPCC intermediate through stabilizing the more formal thiolate.…”

“…The latter interacts electrostatically with CysCT, stabilizing the thiolate-FAD-CT interaction. By contrast, the hydrophobic F501 side chain in 2-KPCC stabilizes the CysCT (C87) thiol (CysCT-SH), so that a CT is only observed at very high pH (pKa = 9.4 for the C82A variant) (5). At neutral pH, the reactive form of 2-KPCC has a single proton in its active site, which is shared between the CysINT (C82) and CysCT (C87) and fully transferred to the latter immediately prior to the reaction with 2-KPC (Scheme 1C) (5).…”

“…A control sample that lacked BES was run in parallel. The samples were allowed to incubate at 20°C for 4 h. This incubation time was previously shown (5) to result in a range of enzyme reactivity with BES, from <10% to nearly stoichiometric, depending on the pH. It was concluded that increasing pH therefore shifts the proton tautomerization state, from a position where it is shared between CysINT/CysCT to CysCT.…”

“…2-ketopropyl-CoM oxidoreductase/carboxylase (2-KPCC) is a member of the well-characterized NAD(P)H/FAD-dependent disulfide oxidoreductase (DSOR) family and a highly unusual bacterial carboxylase (4,5). DSOR enzymes contain a redox-active cysteine pair that catalyzes the two-electron, two-proton reduction of a disulfide substrate (6,7,8), where glutathione disulfide (GSSG), catalyzed by glutathione reductase (GR), is a paradigmatic example (Scheme 1B) (9,10,11,12).…”