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 kcat/Km 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.
Highlights
Pyridine nucleotide disulfide oxidoreductases (PNDORs) are a large class of homodimeric NADH- and FAD-dependent enzymes that generally function to reduce small molecule substrates at a catalytic cysteine positioned at the si face of the isoalloxazine ring
While we have reported the values obtained from these assays, due to the nature of the assay, these rates are sufficiently inaccurate that direct comparison of them to the standard assay would likely result in overinterpretation of the results—they are meant more to serve as qualitative assays confirming the lack of NADH oxidase activity and presence of per/polysulfide reductase activity under anaerobic conditions
Its rhodanese tail resembles the domain found in S. loihica NAD(P)H-dependent persulfide reductase (Npsr) but is absent from CoA-disulfide reductase (CoADR)
Summary
Pyridine nucleotide disulfide oxidoreductases (PNDORs) are a large class of homodimeric NADH- and FAD-dependent enzymes that generally function to reduce small molecule substrates at a catalytic cysteine positioned at the si face of the isoalloxazine ring. The substrate preference and reactivity vary greatly depending on surrounding catalytic residues and additional structural elements, but broadly speaking, each subunit contributes catalytic residues to the active site. Two closely related PNDORs, CoA-disulfide reductase (CoADR) and NAD(P)H-dependent persulfide reductase (Npsr), reduce S0 and facilitate dissimilatory sulfur respiration in prokaryotes and archaea by catalyzing the reduction of either elemental sulfur, persulfides, or polysulfide to hydrogen sulfide. There is a strong correlation between the presence of genes for these proteins and the ability of bacterial and archaeal species to respire by carrying out in vivo S0 Archaea NAD(P)+ NAD(P)H ON HN O R N N SH Cys
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