Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

Abstract

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

Metabolic and Structural Insights into Hydrogen Sulfide Mis-Regulation in Enterococcus faecalis

“…These MD simulations provide significant support for the “swinging pantothenate arm” hypothesis [ 57 ], where the tightly bound CoA attacks a persulfide on C508, bringing CoASSH back to the reductase active site; the CoASSH is then attacked by the C42 with the release of the H 2 S and formation of mixed disulfide, which is subsequently reduced by the flavin, which itself is reduced by a more weakly bound NAD(P)H [ 74 ]. Significant levels of C508 persulfidation in cells on par with those of candidate persulfidation targets Pta and FabN ( Figure 2 ), coupled with no observable C42 persulfidation, are fully consistent with this mechanistic model.…”

“…The Ef CoAPR structure is similar to other FAD-dependent pyridine nucleotide-disulfide oxidoreductases, including those classified as CDRs, NADH (per)oxidases, NAD-dependent persulfide reductases (Npsr) [ 73 , 74 ] and Bacillus anthracis CoADR-RHD ( Ba CoADR-RHD, 3ICS) [ 42 ]. In striking contrast to Ef CoAPR, the entire CoA molecule is visible in these three structures, with the CoA sulfur atom in close proximity to the flavin isoalloxazine ring of the opposite protomer [ 42 , 74 , 75 ]. The high structural similarity between Ef CoAPR and Ba CoADR-RHD models revealed nearly perfect superposition of the PAP moiety of CoA and FAD cofactors ( Supplemental Figure S6A ).…”