NADH‐dependent persulfide reductase [flavoprotein/rhodanase]. S° or polysulfide → sulfide

MKKILIIGGVAGGASAAARARRLSETAEIIMFERGEYVSFANCGLPYHISGEIAQRSALV LQTPESFKARFNVEVRVKHEVVAIDRAAKLVTVRRLLDGSEYQESYDTLLLSPGAAPIVP PIPGVDNPLTHSLRNIPDMDRILQTIQMNNVEHATVVGGGFIGLEMMESLHHLGIKTTLL ELADQVMTPVDREMAGFAHQAIRDQGVDLRLGTALSEVSYQVQTHVASDAAGEDTAHQHI KGHLSLTLSNGELLETDLLIMAIGVRPETQLARDAGLAIGELGGIKVNAMMQTSDPAIYA VGDAVEEQDFVTGQACLVPLAGPANRQGRMAADNMFGREERYQGTQGTAICKVFDLAVGA TGKNEKQLKQAGIAFEKVYVHTASHASYYPGAEVVSFKLLFDPVKGTIFGAQAVGKDGID KRIDVMAVAQRAGMTVEQLQHLELSYAPPYGSAKDVINQAAFVASNIIKGDATPIHFDQI DNLSEDQLLLDVRNPGELQNGGLEGAVNIPVDELRDRMHELPKDKEIIIFCQVGLRGNVA YRQLVNNGYRARNLIGGYRTYKFASV575

	PMID	Title & Author	Abstract	Year
0	24981797	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.Kyu Hyun Lee , Scott Humbarger , Raj Bahnvadia , Matthew H Sazinsky , Edward J Crane 3rd	The NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4 is a member of the single cysteine-containing subset of the family of disulfide reductases represented by glutathione reductase. We have determined the kinetics of the reductive half-reaction of the enzyme with NADH using stopped-flow spectroscopy and kinetic isotope effects, and these results indicate that the reductive and oxidative half-reactions are both partially rate-limiting for enzyme turnover. During reaction with NADH, the reduced nucleotide appears to bind rapidly in an unproductive conformation, followed by the formation of a productive E·NADH complex and subsequent electron transfer to FAD. F161 of Npsr fills the space in which the nicotinamide ring of NADH would be expected to bind. We have shown that while this residue is not absolutely required for catalysis, it does assist in the forward commitment to catalysis through its role in the reductive half reaction, where it appears to enhance hydride transfer in the productive E·NADH complex. While the fluorescence and absorbance spectra of the stable redox forms of the wild-type and F161A mutant enzymes are similar, intermediates formed during reduction and turnover have different characteristics and appear to indicate that the enzyme-NADH complex formed just prior to hydride transfer on the F161A enzyme has weaker FAD-NADH interactions than the wild-type enzyme, consistent with a "looser" enzyme-NADH complex. The 2.7Å crystal structure of the F161A mutant was determined, and shows that the nicotinamide ring of NADH would have the expected freedom of motion in the more open NADH binding cavity.	2014
1	21090815	Characterization of an NADH-dependent persulfide reductase from Shewanella loihica PV-4: implications for the mechanism of sulfur respiration via FAD-dependent enzymes.Megan D Warner , Vinita Lukose, Kyu Hyun Lee, Karlo Lopez, Matthew H Sazinsky, Edward J Crane 3rd	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.	2011

Warner, M.D., Lukose, V., Lee, K.H., Lopez, K., Sazinsky, M.H., and Crane, E.J. 3rd. (2011) Characterization of an NADH‐dependent persulfide reductase from Shewanella loihica PV‐4: implications for the mechanism of sulfur respiration via FAD‐dependent enzymes. Biochemistry 50: 194–206.