Characterization of an NADH-Dependent Persulfide Reductase from Shewanella loihica PV-4: Implications for the Mechanism of Sulfur Respiration via FAD-Dependent Enzymes^,

Abstract

The NADH-dependent persulfide reductase (Npsr), a recently discovered member of the PNDOR family of flavoproteins that contains both the canonical flavoprotein reductase domain and a rhodanese domain, is proposed to be involved in the dissimilatory reduction of S(0) for Shewanella loihica PV-4. We have previously shown that polysulfide is a substrate for this enzyme, and a recently determined structure of a closely related enzyme (CoADR-Rhod from Bacillus anthracis) suggested the importance of a bound coenzyme A in the mechanism. The work described here shows that the in vivo oxidizing substrates of Npsr are the persulfides of small thiols such as CoA and glutathione. C43S, C531S, and C43,531S mutants were created to determine the role of the flavoprotein domain cysteine (C43) and the rhodanese domain cysteine (C531) in the mechanism. The absolute requirement for C43 in persulfide or DTNB reductase activity shows that this residue is involved in S-S bond breakage. C531 contributes to, but is not required for, catalysis of DTNB reduction, while it is absolutely required for reduction of any persulfide substrates. Titrations of the enzyme with NADH, dithionite, titanium(III), or TCEP demonstrate the presence of a mixed-disulfide between C43 and a tightly bound CoA, and structures of the C43 and C43,531S mutants confirm that this coenzyme A remains tightly bound to the enzyme in the absence of a C43-CoA S-S bond. The structure of Npsr suggests a likely site for binding and reaction with the persulfide substrate on the rhodanese domain. On the basis of kinetic, titration, and structural data, a mechanism for the reduction of persulfides by Npsr is proposed.

Novel role of 4-hydroxy-2-nonenal in AIFm2-mediated mitochondrial stress signaling

“…The NADH-dependent persulfide reductase (Npsr) from S. loihica (PDB=3NTA) [35] was utilized as the template structure. It has 17% sequence identity and 96% backbone coverage, resulting in a model with 100% fold probability confidence.…”

Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: Formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes

“…The F161A mutant shows even less long wavelength absorbance than the wild-type enzyme in the EH 2 and EH 2 /EH 4 forms (Fig. 2) [5]. The difference in the apparent equivalents of NADH consumed between the wild-type and F161A enzymes could be explained by either 1) a lack of NADH binding to the EH 2 form of the enzyme, or by 2) different spectral characteristics of any EH 2 ·NADH complex that does form (such as an increased A 340 ).…”

“…1 ± 0.2 s −1 ) to a rate for deuteride ( 2 H − ) transfer close to that of other rate-limiting step(s), most likely in the oxidative half-reaction, during reaction with (4S)-[4-2 H]NADH and DTNB (k 2 = 6.98-18.8 s −1 vs. a k cat of 7.02 s −1 ). For reaction with the most efficient physiological substrate, CoA persulfide [5], the overall reaction rate is likely to be controlled by both the oxidative and reductive half reactions, as k cat for that substrate is roughly 3-fold higher than that for DTNB.…”

“…This means that ratelimiting flavin reduction is followed by very rapid oxidation of the reduced flavin by the disulfide: the FADH − /FADH 2 species is not observed because it is oxidized immediately after formation. When modeling this reaction via Kinsim [28] as a 4-step reaction, as shown in Table 4 and Scheme 1, the rate of disulfide reduction by the FADH 2 /FADH − intermediate (with the extinction coefficient of dithionite-reduced Npsr [5]) indicates that the rate of FAD oxidation (k 4 ) must occur at~2000 s −1 in order for the reduced FAD species to be effectively "hidden" in this manner. In Fig.…”

“…Npsr reacts with disulfide, persulfide and polysulfide substrates via a single redox active site cysteine and a tightly bound coenzyme A. The bound CoA has been proposed to shuttle sulfide equivalents between a surface cysteine, located on a rhodanase-like domain, through the substrate channel to the active site CoA/cys disulfide [5].…”