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Last modified: 11 November 1998

Sulphite oxidase family
Domain Prosthetic group Formal oxidation states
Moco
Mo cofactor image

Mo·molybdopterin (R = H)
MoIV; MoV; MoVI
High pH form
Low pH form
2oxo-Molybdopterin-Cys image

[MoO2](Spterin)2SgammaCys
2oxo-Molybdopterin-Cys image

[MoO2](Spterin)2SgammaCys
MoVI
oxo, hydroxo-Molybdopterin-Cys image

[MoOOH](Spterin)2SgammaCys
oxo, hydroxo, X-Molybdopterin-Cys image

[MoOOHX](Spterin)2SgammaCys
(X = Cl or S)
MoIV; MoV
Cyt b Haem type Haem iron coordination Axial iron ligands Formal iron oxidation states
Haem b image
Haem b
His-Haem-His image
Hexacoordinate
NepsilonHis;

NepsilonHis

FeII; FeIII
FAD Prosthetic group Oxidation states
FAD image

Flavin adenine dinucleotide (FAD)
FAD;

FAD· (semiquinone);

FADH2

Enzymes of the sulphite oxidase family contain a molybdenum cofactor (Moco) and catalyse reactions that involve the oxygen atom transfer to or from an available electron lone pair of a substrate (1, 2). These enzymes possess a dioxo­Mo centre, with most likely one molybdopterin coordinated to the Mo via the dithiolene sulphur atoms [1-3].

Sulphite oxidase catalyses the oxidation of sulphite to sulphate (1), with ferricytochrome c as physiological electron acceptor.

The enzyme usually exists as a homodimer, each monomer consisting of two functional domains. The smaller N­terminal domain binds haem and is homologous to cytochrome b5; the larger C­terminal domain binds the Moco. Tryptic cleavage of rat liver sulphite oxidase yields two fragments corresponding to these domains. The isolated cyt b5 domain has no sulphite oxidase activity, but can mediate electron transfer between NADH:cyt b5 reductase and cyt c. The isolated Moco domain has no sulphite:ferricyt c activity, but retains sulphite:ferricyanide and sulphite:O2 activity [4]. The electron flow in the sulphite oxidase reaction is The first step of biological nitrate assimilation, reduction of nitrate to nitrite (2) is catalysed by the multidomain redox enzyme nitrate reductase (NR). Three forms of NR are known: an NADH­specific enzyme found in higher plants and algae (EC 1.6.6.1); an NAD(P)H­bispecific enzyme found in higher plants, algae and fungi (EC 1.6.6.2); and an NADPH­specific enzyme found only in fungi (EC 1.6.6.3) [5]. The reaction (2) is thought to be essentially the reverse of reaction (1) due to the relative stabilities of the N=O and S=O bonds (nitrate is an effective oxo donor while sulphite is a more effective oxo acceptor) [3]. The enzyme usually exists as a homodimer, each monomer consisting of three domains. The Moco binds in the N­terminal domain homologous to the C­terminal domain of sulphite oxidase; the central region is the cytochrome b5­like domain (cyt b557); and the C­terminal portion of the protein is occupied by the FAD/NAD(P)H binding domain, which is similar to NADH:cytochrome b5 reductase (CbR). The catalytic reduction of nitrate to nitrite can be viewed as a single polypeptide electron transport chain with electron flow Moco­binding domains of sulphite oxidase and NRs are highly similar and form a distinct family [6]. Despite functional parallels, members of the family show no sequence similarity to the C­terminal molybdopterin domain of xanthine dehydrogenase. Comparison of the sequences of the Moco­binding domains of sulphite oxidase and NRs suggests that only a single Cys residue (Cys­207 in rat and human sulphite oxidase, Cys­185 in chicken sulphite oxidase, Cys­150 in Aspergillus nidulans NR), is invariant in all these enzymes, indicating that it plays a role in binding Moco to the protein [7, 8]. Site­directed mutagenesis studies of this residue showed that it is essential for enzyme activity; the spectral changes suggest that Cys­207 functions as a ligand of the Mo [9].

The crystal structures of chicken liver sulphite oxidase [10] and the CbR (flavin) domain of maize NR [11] have been solved. Sulphite oxidase is a homodimer, each monomer consisting of three structural domains. The N­terminal domain (residues 3-84) is similar to cytochrome b5, with haem iron symmetrically coordinated by His­40 and His­65. Domain II (residues 96-323) represents a novel alpha+ß fold containing 9 alpha­helices and 13 ß­strands organised in three ß­sheets. The Moco is bound at the centre of this domain and is contacted by discontinuous stretches of the polypeptide chain. The core of the domain III (residues 324-466) has the C2 subtype immunoglobulin­like fold (seven ß­strands organised in two antiparallel ß­sheets). In addition, domain III contains two long loops with two short ß­helices. Domains III form the dimerisation interface in a head­to­head arrangement. Note that functional C­terminal domain yielded by proteolysis (see above) corresponds to the two structural domains, II and III. The CbR domain of NR shares a fold with other members of the ferredoxin:NADP+ reductase (FNR) family: the N­terminal FAD­binding subdomain (residues 1-119), which has an antiparallel ß­barrel topology, and the C­terminal NADH­binding subdomain (residues 136-270) with ßalphaß structure. A deep cleft is formed between the two subdomains where the two cofactors can bind [11].

The Moco consists of a single molybdopterin and Mo ion. Structural information on coordination of Mo has been provided by EXAFS, EPR and resonance Raman spectroscopies. EXAFS studies [12] show that in oxidised sulphite oxidase Mo is pentacoordinated, with two oxygen and three thiolate ligands. The coordination of the Mo is sensitive to the oxidation state of the metal and to the pH of the solution: at pH 9.0, the Mo in all three oxidation states has five ligands; at pH 6.0, an additional ligand (sulphur or chloride) is observed in the MoIV and MoV states. It has been suggested that the Mo has either a square pyramidal or an octahedral coordination geometry with one oxygen and three thiolates as the equatorial ligands, the second oxo group occupying one axial position, and the remaining ligand (present only in low pH MoIV and MoV forms) at the second axial position [1]. Resonance Raman studies also indicate that two cis­oxo groups are present in oxidised sulphite oxidase [13]. In the crystal structure, the Mo has a square pyramidal coordination geometry with the terminal oxo group occupying the axial position (Mo-O distance 1.7 Å) and one water/hydroxo oxygen and three thiolates as the equatorial ligands. The thiolate ligands are the two dithiolene sulphurs of the molybdopterin (Mo-S distances 2.4 Å) and Sgamma of Cys­185 (Mo-S distance 2.5 Å). The fourth equatorial ligand was assigned as a water or hydroxide due to the long Mo-O distance (2.3 Å). This coordination was unexpected since the protein was purified in its fully oxidised (MoVI/FeIII) form. However, the authors argue that the oxidised enzyme might have been reduced in the crystal [10]. Adjacent to the water/hydroxo ligand, a bound molecule of sulphate or sulphite was observed.

Mo cofactor - SO4 complex image

Sulphite oxidase family in enzyme databases

ENZYME LIGAND BRENDA Official name Alternative name
1.6.6.1 1.6.6.1 1.6.6.1 Nitrate reductase (NADH) Assimilatory nitrate reductase
1.6.6.2 1.6.6.2 1.6.6.2 Nitrate reductase [NAD(P)H] Assimilatory nitrate reductase
1.6.6.3 1.6.6.3 1.6.6.3 Nitrate reductase (NADPH) Assimilatory nitrate reductase
1.8.3.1 1.8.3.1 1.8.3.1 Sulphite oxidase Sulphite:ferricytochrome c oxidoreductase

Sulphite oxidase family in motif databases

PRINTS ID PRINTS AC PROSITE/BLOCKS ID PROSITE AC BLOCKS AC
EUMOPTERIN PR00407 MOLYBDOPTERIN_EUK PS00559 BL00559
CYTOCHROMEB5 PR00363 CYTOCHROME_B5 PS00191 BL00191
CYTB5RDTASE PR00406
-
-
-

Sulphite oxidase family in alignment databases

Protein Family Protein Homology Domain Pfam LPFC 3­D
alignment
00132; nitrate reductase
00143; sulfite oxidase 		00259; molybdopterin­binding domain PF00174; oxidored_molyb
-
00006; cytochrome b5 core PF00173; heme_1
-
00050; cytochrome b5 reductase PF00175; oxidored_fad
-

Sulphite oxidase family in 3­D databases

NADH:nitrate reductase contains a Moco (molybdopterin­binding domain), haem b (cytochrome b5­like domain) and FAD (cytochrome b reductase domain); sulphite oxidase contains a Moco (molybdopterin­binding domain) and haem b (cytochrome b5­like domain).

PDB scop BSM RELI
Base		 Header MMS Abstract ¹
1cne 1cne 1cne 1cne NADH:nitrate reductase (cytochrome b reductase fragment) (C242S mutant); maize (Zea mays)
-
1cnf 1cnf 1cnf 1cnf NADH:nitrate reductase (cytochrome b reductase fragment) (complex with ADP); maize (Zea mays)
-
1sox
-
 1sox 1sox Sulphite oxidase (complex with SO42+, glycerol and HEPES); chicken (liver)
-
2cnd 2cnd 2cnd 2cnd NADH:nitrate reductase (cytochrome b reductase fragment); maize (Zea mays) MS5PM6

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

  1. Kisker, C., Schindelin, H. and Rees, D.C. (1997) Molybdenum­cofactor-containing enzymes: structure and mechanism. Annu. Rev. Biochem. 66, 233-267.
  2. Hille, R. (1996a) Structure and function of mononuclear molybdenum enzymes. J. Biol. Inorg. Chem. 1, 397-404.
  3. Hille, R. (1996b) The mononuclear molybdenum enzymes. Chem. Rev. 96, 2757-2816.
  4. Johnson, J.L. and Rajagopalan, K.V. (1977) Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains. J. Biol. Chem. 252, 2017-2025.
  5. Campbell, W.H. and Kinghorn, K.R. (1990) Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem. Sci. 15, 315-319.
  6. Wootton, J.C., Nicolson, R.E., Cock, J.M., Walters, D.E., Burke, J.F., Doyle, W.A. and Bray, R.C. (1991) Enzymes depending on the pterin molybdenum cofactor: Sequence families, spectroscopic properties of molybdenum and possible cofactor­binding domains. Biochim. Biophys. Acta 1057, 157-185.
  7. Barber, M.J. and Neame, P.J. (1990) A conserved cysteine in molybdenum oxotransferases. J. Biol. Chem. 265, 20912-20915.
  8. Garde, J., Kinghorn, J.R. and Tomsett, A.B. (1995) Site­directed mutagenesis of nitrate reductase from Aspergillus nidulans. Identification of some essential and some nonessential amino acids among conserved residues. J. Biol. Chem. 270, 6644-6650.
  9. Garrett, R.M. and Rajagopalan, K.V. (1996) Site­directed mutagenesis of recombinant sulfite oxidase: Identification of cysteine 207 as a ligand of molybdenum. J. Biol. Chem. 271, 7387-7391.
  10. Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W.A., Garrett, R.M., Rajagopalan, K.V., Enemark, J.H. and Rees, D.C. (1997) Molecular basis of sulfite oxidase deficiency from the structure of sulfite oxidase. Cell 91, 973-983.
  11. Lu, G., Lindqvist, Y., Schneider, G., Dwivedi, U. and Campbell, W.H. (1995) Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 Å, its ADP complex and an active­site mutant and modeling of the cytochrome b domain. J. Mol. Biol. 248, 931-948.
  12. George, G.N., Kipke, C.A., Prince, R.C., Sunde, R.A., Enemark, J.H. and Cramer, S.P. (1989) Structure of the active site of sulfite oxidase. X­ray absorption spectroscopy of the Mo(IV), Mo(V), and Mo(VI) oxidation states. Biochemistry 28, 5075-5080.
  13. Garton, S.G., Garrett, R.M., Rajagopalan, K.V. and Johnson, M.K. (1997) Resonance Raman characterization of the molybdenum center in sulfite oxidase: Identification of Mo=O stretching modes. J. Am. Chem. Soc. 119, 2590-2591.
Bibliography on structural studies of sulphite oxidase family