TSRI's main web site PROMISE mirror at TSRI Metalloprotein DB site Created: 13 May 1998
Last modified: 26 November 1998

Hydroxylamine oxidoreductase
Haem Haem type Haem iron coordination Axial iron ligands Formal iron oxidation/spin states
4 (P460)
Haem P460 image
Haem P460
Haem-His image
Pentacoordinate
NepsilonHis
 FeII (S=2);

FeIII (S=5/2 or S=3/2)|

| Spin­coupled to haem 6
His-haem-CO image
Hexacoordinate
NepsilonHis;

substrate, CO, NO, CN¯ or other ligand

FeII (S=0);

FeIII (S=1/2)
1

2

3

5

6

7

8

Haem c image
Haem c
His-Haem-His image
Hexacoordinate
NepsilonHis;

NepsilonHis

FeII (S=0);

FeIII (S=1/2)

Haem 6 is spin­coupled to haem P460

Hydroxylamine oxidoreductase (HAO; EC 1.7.3.4) catalyses the four­electron oxidation of hydroxylamine to nitrite (1). HAO is one of several abundant periplasmic cytochromes c (cyt c) in a nitrifying chemoautotrophic bacterium Nitrosomonas europaea and is a key enzyme in the respiratory chain [1].

Each subunit of the homotrimeric enzyme contains eight covalently bound haem groups, designated 1 to 8 according to the position of the haem binding motif CxxCH in the sequence. These are the seven haems c and an unusual haem P460 (haem 4). Haem P460 has a Soret band at 463 nm in the fully reduced form and binds CO. This suggests that the P460 is a high­spin pentacoordinated haem and appears to be involved in the catalysis, while the haem c groups participate in electron transfer to the electron acceptor, cytochrome c554. Some properties of the HAO haems are summarised in the table:

Group Haem Em (mV) [2] Haem iron ligands Covalent links Absorption
spectral
features (nm)
pH 6 pH 7 pH 8 Proximal Distal Soret alpha
I 4 (P460) -203 -260 -322 His­233 (substrate) Cys­229; Cys­232; Tyr­467 (adjacent subunit) 463 560
6 -190 -192 -192 His­263 His­204 Cys­259; Cys­262 420 553
7 -265 -265 -269 His­314 His­459 Cys­310; Cys­313 420 553
II 3 -9 -10 -16 His­176 His­99 Cys­172; Cys­175 420 553
5 -138 -162 -151 His­243 His­323 Cys­239; Cys­242 420 553
III 1 +299 +288 +259 His­83 His­160 Cys­79; Cys­82 420 553
2 +30 +11 +18 His­149 His­246 Cys­145; Cys­148 420 559
IV 8 -384 -412 -402 His­364 His­279 Cys­360; Cys­363 420 553

The 3­D structure of HAO has been determined [3]. The molecule exists as a trimer (100 diam.×100 Å) with the subunits arranged as cloves in a head of garlic. Each subunit is folded into two domains (the flexible C­terminal domain is not resolved). The N­terminal domain (residues 1-269) contains a short two­stranded ß­sheet and 14 alpha­helices, with five haems c and haem P460. The central domain (residues 270-499) contains ten alpha­helices and the two remaining haems, 7 and 8.

All haems c are bis­His coordinated, while haem P460 is pentacoordinated, with His­233 as an axial iron ligand (see table for details). The haem P460, as the haems c, is covalently attached to the protein via two thioether bonds (Cys­229 and Cys­232). An additional covalent bond exists between haem meso­carbon and Cepsilon of Tyr­467 from the adjacent subunit. Within each monomer, the haems are clustered into four groups. Group I (triple haem cluster) consists of haems P460, 6 and 7, stacked parallel to each other. Haem 6 appears to be exchange­coupled to the P460 haem [4]. In the two double haem clusters, called group II (haems 3 and 5) and group III (haems 1 and 2), haems are also parallel to each other. Haem 5 also interacts with haem 6 of the triple haem cluster. Group IV includes the single haem 8, which may interact with haem 7 and group III of an adjacent subunit [3]. The 3­D structure of cytochrome c554 reveals the striking structural similarities between the tetrahaem core of cytochrome c554 and haem groups 3 to 6 of HAO [5].

It has been suggested that the Tyr residue attached to the P460 and the arrangement of haem clusters may allow HAO to transiently accept two electrons at the same time. Thus reaction (1) may occur in two two­electron steps:


Hydroxylamine oxidoreductase in enzyme databases

ENZYME LIGAND BRENDA Official name Alternative names
1.7.3.4
1.7.3.4
1.7.3.4
Hydroxylamine oxidase
Hydroxylamine oxidoreductase; HAO

Hydroxylamine oxidoreductase in motif databases

PRINTS ID PRINTS AC PROSITE/BLOCKS ID PROSITE AC BLOCKS AC
-
-
 CYTOCHROME_C PS00190 BL00190

Hydroxylamine oxidoreductase in SWISS­PROT/TREMBL

HAO_NITEU Hydroxylamine oxidoreductase precursor; Nitrosomonas europaea

Hydroxylamine oxidoreductase in alignment databases

Protein Family Pfam LPFC 3­D alignment
22197; hydroxylamine oxidase
-
-

Hydroxylamine oxidoreductase in 3­D databases

HAO contains seven covalently bound haems c and one covalently bound haem P460.

PDB MSD scop BSM RELI
Base		 Header MMS Abstract ¹
1fgj 1fgj
-
 1fgj 1fgj Hydroxylamine oxidoreductase; Nitrosomonas europaea
-

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

References

  1. Prince, R.C. and George, G.N. (1997) The remarkable complexity of hydroxylamine oxidoreductase. Nature Struct. Biol. 4, 247-250.
  2. Collins, M.J., Arciero, D.M. and Hooper, A.B. (1993) Optical spectropotentiometric resolution of the hemes of hydroxylamine oxidoreductase. Heme quantitation and pH dependence of Em. J. Biol. Chem. 268, 14655-14662.
  3. Igarashi, N., Moriyama, H., Fujiwara, T., Fukumori, Y. and Tanaka, N. (1997) The 2.8 Å structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea. Nature Struct. Biol. 4, 276-284.
  4. Arciero, D.M., Golombek, A., Hendrich, M.P. and Hooper, A.B. (1998) Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 37, 523-529.
  5. Iverson, T.M., Arciero, D.M., Hsu, B.T., Logan, M.S.P., Hooper, A.B. and Rees, D.C. (1998) Heme packing motifs revealed by the crystal structure of the tetra­heme cytochrome c554 from Nitrosomonas europaea. Nature Struct. Biol. 5, 1005-1012.
Bibliography on structural studies of hydroxylamine oxidoreductase