NADH peroxidase

 

NADH peroxidase (Npx) catalysis the breakdown of peroxide to water. It is unusual in two respects, having a redox centre containing a key sulfenic acid and requiring NADH for activity. The critical and defining property of Npx relative to other FAD-dependent disulfide reductases concerns the absence of the redox-active protein disulfide.

 

Reference Protein and Structure

Sequence
P37062 UniProt (1.11.1.1) IPR023753 (Sequence Homologues) (PDB Homologues)
Biological species
Enterococcus faecalis V583 (Streptococcus faecalis) Uniprot
PDB
2npx - NADH BINDING SITE AND CATALYSIS OF NADH PEROXIDASE (2.4 Å) PDBe PDBsum 2npx
Catalytic CATH Domains
3.50.50.60 CATHdb (see all for 2npx)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.11.1.1)

hydrogen peroxide
CHEBI:16240ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
→
water
CHEBI:15377ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
Alternative enzyme names: DPNH peroxidase, NAD peroxidase, NADH-peroxidase, Diphosphopyridine nucleotide peroxidase, Nicotinamide adenine dinucleotide peroxidase,

Enzyme Mechanism

Introduction

Initially, Cys42 is present as the thiolate S- ion which is catalytically competent. The peroxide binds to the active site and is predicted to hydrogen bond via an oxygen to the His10 N-H group. As the thiolate anion performs nucleophilic attack on the O-O bond of peroxide, His10 donates a proton the the leaving hydroxide to form water. Simultaneously a proton is donated by Arg303 to the second His10 nitrogen maintaining its neutral charge. As a result sulfenic acid is formed, which is then reduced to complete the catalytic cycle.

NADH binds parallel to FAD on the re face with C4 in a position such that a direct hydride transfer to N5 of FAD occurs. FAD is thus reduced to FADH2 with the aid of a proton. A hydride is then transferred to Cys-SOH, which lies parallel to the si face of FAD, causing lysis of the S-O bond and formation of a second water and thiolate, returning the enzyme to its initial state.

Catalytic Residues Roles

UniProt PDB* (2npx)
His10 His10A Forms a weak hydrogen bond to Cys-SOH causing deprotonation and thus increasing Cys42 nucleophilicity. Forms a hydrogen bond to the oxygen of peroxide then donates a proton as Cys42 attacks. proton shuttle (general acid/base)
Cys42 (ptm) Ocs42A (ptm) Cys42 acts as the neuceophile in this reaction. covalent catalysis, proton shuttle (general acid/base)
Ser41 (main-N) Ser41A (main-N) Helps stabilise the reactive intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Arg303 Arg303A Donates a proton to His10 during the Cys42 nucleophilic attack thus maintaining His10's neutrality. proton shuttle (general acid/base)
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Yeh JI et al. (1996), Biochemistry, 35, 9951-9957. Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution. DOI:10.1021/bi961037s. PMID:8756456.
  2. Wallen JR et al. (2015), Biochemistry, 54, 6815-6829. Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate. DOI:10.1021/acs.biochem.5b00676. PMID:26506002.
  3. Yeh JI et al. (2002), Methods Enzymol, 353, 44-54. Crystal structures of oxidized and reduced forms of NADH peroxidase. PMID:12078517.
  4. Claiborne A et al. (2001), Adv Protein Chem, 58, 215-276. Structural, redox, and mechanistic parameters for cysteine-sulfenic acid function in catalysis and regulation. DOI:10.1016/s0065-3233(01)58006-7. PMID:11665489.
  5. Crane EJ 3rd et al. (2000), Biochemistry, 39, 10353-10364. Analysis of the Kinetic and Redox Properties of the NADH Peroxidase R303M Mutant:  Correlation with the Crystal Structure†,‡. DOI:10.1021/bi000553m. PMID:10956025.
  6. Claiborne A et al. (1999), Biochemistry, 38, 15407-15416. Protein-Sulfenic Acids:  Diverse Roles for an Unlikely Player in Enzyme Catalysis and Redox Regulation†. DOI:10.1021/bi992025k. PMID:10569923.
  7. Mallett TC et al. (1999), Biochemistry, 38, 3000-3011. Equilibrium analyses of the active-site asymmetry in enterococcal NADH oxidase: role of the cysteine-sulfenic acid redox center. DOI:10.1021/bi9817717. PMID:10074352.
  8. Mallett TC et al. (1998), Biochemistry, 37, 8790-8802. Oxygen reactivity of an NADH oxidase C42S mutant: evidence for a C(4a)-peroxyflavin intermediate and a rate-limiting conformational change. DOI:10.1021/bi9803630. PMID:9628741.
  9. Crane EJ 3rd et al. (1997), Biochemistry, 36, 8611-8618. 13C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase. DOI:10.1021/bi9707990. PMID:9214307.
  10. Crane EJ 3rd et al. (1996), Biochemistry, 35, 2380-2387. The Active-Site Histidine-10 of Enterococcal NADH Peroxidase Is Not Essential for Catalytic Activity†. DOI:10.1021/bi952347y. PMID:8652580.
  11. Mande SS et al. (1995), Biochemistry, 34, 6985-6992. Crystallographic Analyses of NADH Peroxidase Cys42Ala and Cys42Ser Mutants: Active Site Structures, Mechanistic Implications, and an Unusual Environment of Arg 303. DOI:10.1021/bi00021a010. PMID:7766608.
  12. Parsonage D et al. (1995), Biochemistry, 34, 435-441. Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center. DOI:10.1021/bi00002a007.
  13. Marcinkeviciene JA et al. (1995), Biochemistry, 34, 6621-6627. Quinone reductase reaction catalyzed by Streptococcus faecalis NADH peroxidase. PMID:7756294.
  14. Miller H et al. (1995), Biochemistry, 34, 5180-5190. An L40C mutation converts the cysteine-sulfenic acid redox center in enterococcal NADH peroxidase to a disulfide. PMID:7711038.
  15. Stehle T et al. (1993), Eur J Biochem, 211, 221-226. NADH binding site and catalysis of NADH peroxidase. DOI:10.1111/j.1432-1033.1993.tb19889.x. PMID:8425532.
  16. Stehle T et al. (1991), J Mol Biol, 221, 1325-1344. Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution. PMID:1942054.

Step 1.

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Catalytic Residues Roles

Residue Roles
Ocs42A (ptm) covalent catalysis
His10A proton shuttle (general acid/base)
Ocs42A (ptm) proton shuttle (general acid/base)
Arg303A proton shuttle (general acid/base)
Ser41A (main-N) electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Craig Porter, Gemma L. Holliday