Biochemical Society Transactions

Biochemical Society Focused Meeting

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria

Margreet J. Oosterkamp, Farrakh Mehboob, Gosse Schraa, Caroline M. Plugge, Alfons J.M. Stams


The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.

  • chlorite dismutase
  • molybdenum-containing enzyme
  • nitrate reductase
  • nitrate reduction
  • (per)chlorate reductase
  • (per)chlorate reduction


Perchlorate and chlorate, termed (per)chlorate, are compounds that are used as solid rocket fuel and in road flares, fireworks, matches, blasting agents, explosives and lubricating oils [13]. Besides their anthropogenic origin, (per)chlorate may also occur in the environment by natural production [46]. Natural deposits of large amounts of (per)chlorate can be found in the hyper-arid region of the Atacama desert of Chile [7]. Owing to their high solubility, (per)chlorate salts are readily transported and can be detected in surface water and groundwater [3,8]. Bacteria are able to use (per)chlorate as a terminal electron acceptor for growth [9,10]. Characteristically, molecular oxygen is produced upon (per)chlorate reduction [9,11]. The microbial reduction of perchlorate proceeds as follows: Embedded Image

Perchlorate (ClO4) is reduced by perchlorate reductase to chlorate (ClO3), which in turn is reduced to chlorite (ClO2) by chlorate reductase. In (per)chlorate-reducing bacteria, one enzyme may reduce both perchlorate and chlorate [12]. Chlorite is then split into Cl and O2 by a chlorite dismutase [9,10]. Recently, oxygen formation was also found in a culture that degraded methane anaerobically with nitrite. The proposed pathway involves a yet hypothetical ‘NO dismutase’, which, like chlorite dismutase, releases the molecular oxygen [13]. Oxygen produced in the (per)chlorate pathway is proposed to be utilized for biodegradation of anaerobically recalcitrant compounds, such as aromatic hydrocarbons [11,14,15]. In this respect, it is highly important to study the mechanisms involved in anaerobic (per)chlorate and nitrate reduction. Nitrate can also be used for bioremediation in anaerobic environments. Dechloromonas aromatica is a (per)chlorate-reducing bacterium that has been described to degrade benzene with nitrate [16]. The previously described Alicycliphilus denitrificans also degrades benzene with chlorate, but not with nitrate [15]. In the present review, we compare (per)chlorate and nitrate reduction in (per)chlorate-reducing micro-organisms.

(Per)chlorate reductase

(Per)chlorate reductases belong to the type II DMSO family of enzymes [17,18]. Type II DMSO reductases have a common Moco (molybdenum cofactor) known as bis(MGD)Mo [bis(molybdopterin guanine dinucleotide)molybdenum] [19]. (Per)chlorate reductases are similar to other type II DMSO reductases, such as nitrate and selenate reductase and ethylbenzene dehydrogenase. (Per)chlorate reductases can also reduce nitrate.

The α-subunit containing the molybdopterin is the catalytic subunit of (per)chlorate reductase. The β-subunit contains the Fe−S cluster and may be involved in electron transfer to the catalytic subunit. The γ-subunit is a cytochrome b moiety of the enzyme. The δ-subunit is not a part of the mature enzyme and is proposed to be a chaperone involved in the assembly of the αβ complex [17,18]. All (per)chlorate reductases known to date are periplasmic. Characteristics of known (per)chlorate reductases are shown in Table 1.

View this table:
Table 1 Characteristics of (per)chlorate reductases

Chlorite dismutase

Chlorite dismutase is a key enzyme in the (per)chlorate reduction pathway. The systematic name of this enzyme should be chloride:oxygen oxidoreductase [21]. It is one of the few oxygen-generating enzymes in Nature and the only one to form an O−O double bond besides Photosystem II. Chlorite dismutase is a haem-containing homotetrameric enzyme, which is present in the periplasm [2225]. Labelling studies confirmed that both atoms of dioxygen originate from chlorite [22,26]. The structural features of the enzyme have been described elsewhere [27].

(Dissimilatory) nitrate reductase

Dissimilatory nitrate reductase is a terminal oxidoreductase that can convert nitrate (NO3) into nitrite (NO2). There are two types of prokaryotic dissimilatory nitrate reductases: the membrane-bound respiratory nitrate reductases and the dissimilatory periplasmic nitrate reductases. In contrast with the membrane-bound nitrate reductase, the periplasmic enzyme cannot reduce chlorate [28]. The periplasmic nitrate reductase is thought to be involved in aerobic denitrification, the transition from aerobiosis to anaerobiosis and the dissipation of an excess reducing power [29]. Since the periplasmic enzyme has only a secondary role in growth under anaerobic conditions and also does not reduce chlorate, this group of nitrate reductases will not be described further.

The common property of all of the nitrate reductases studied is the presence of the Mo factor and Moco in the active centre of the enzyme. Respiratory nitrate reductases are composed of two or three subunits, depending on the enzyme isolation procedure. The enzymes are encoded by the narGHJI operon. The large subunit of the enzymes, the α-subunit, is encoded by narG. The α-subunit carries molybdenum in the form of the MGD cofactor [30,31], which is part of the active site of the enzyme. The narH gene encodes the small or β-subunit which binds Fe−S clusters, three [Fe4−S4] clusters and one [Fe3−S4] cluster, that are part of the active site as well. Respiratory nitrate reductases are anchored to the cytoplasmic membrane by the γ-subunit, encoded by narI. NarJ is not present in the purified enzyme, but it is needed for nitrate reductase biosynthesis. Studies on Escherichia coli respiratory nitrate reductase have shown that NarJ is a system-specific chaperone that stays in the cytosol after assembly of the αβγ complex in the membrane [32]. NarJ was found to be involved in the acquisition of the MGD cofactor by the αβ complex before its attachment to the γ-subunit [33].

Structural similarities and variations of nitrate reductase and (per)chlorate reductase

The (per)chlorate and nitrate reductases both belong to type II DMSO reductases containing a common bis(MGD) cofactor. Unlike the membrane-bound respiratory nitrate reductase, (per)chlorate reductases are periplasmic in nature. With respect to their localization (per)chlorate reductases are more similar to the periplasmic nitrate reductase, but, in contrast, periplasmic nitrate reductases do not show activity with chlorate [28].

Identical with the α-subunit of respiratory nitrate reductase, the α-subunit of (per)chlorate reductase is the catalytic centre and contains the Moco. The size of the α-subunit of (per)chlorate reductase varies from 60 to 100 kDa, whereas the size of the α-subunit of respiratory nitrate reductase is 104–150 kDa [34]. Similarly, identical with the β-subunit of respiratory nitrate reductase, the β-subunit of (per)chlorate reductase contains the Fe–S cluster, which is involved in electron transfer to the catalytic subunit. The size of the β-subunit of (per)chlorate reductase varies from 35 to 63 kDa, which is similar to the size of the β-subunit of respiratory nitrate reductase which is 43–63 kDa [34]. The γ-subunit of respiratory nitrate reductase is a two b-type haem-containing integral membrane protein subunit. The γ-subunit of (per)chlorate reductase is a cytochrome b moiety of the enzyme [20]. The size of this subunit in (per)chlorate reductase ranges from 27 to 56 kDa, whereas this is 19–28 kDa in respiratory nitrate reductase [34]. The γ-subunit of (per)chlorate reductase, unlike NarI, is not a membrane-bound protein [17]. Likewise, similar to the δ-subunit of nitrate reductase, which is necessary for the respiratory nitrate reductase assembly, the δ-subunit of (per)chlorate reductase is a chaperone involved in the assembly of the αβ complex.

To date, no crystal structure of any chlorate reductase is available for comparison with the respiratory nitrate reductase structure.

Similarities and variations of nitrate and (per)chlorate reductase sequences

Just as the αβγδ-subunits of nitrate reductase are encoded by narGHIJ genes, these subunits in (per)chlorate reductase are encoded by clrABCD genes. In Ideonella dechloratans [18] and Pseudomonas chloritidismutans [35] the arrangement of the chlorate reductase genes is clrABDC, which is similar to the arrangement of their nitrate reductase genes, i.e. narGHJI. However, in Dechloromonas agitata and D. aromatica, the order of the genes is pcrABCD [17]. There is high sequence similarity between the genes coding for different subunits of respiratory nitrate reductase and the genes coding for the corresponding subunits in (per)chlorate reductase. Multiple sequence alignment of catalytic subunits of (per)chlorate and nitrate reductases (Figure 1) shows the presence of a highly conserved aspartate residue that acts as a ligand that binds to Mo in respiratory nitrate reductase [36]. Since (per)chlorate reductase is periplasmic, its catalytic subunit has the motif for the Tat (twin-arginine translocation) pathway [17,18,37]. It is suggested that the αβ complex is assembled in the cytoplasm with the help of the δ-subunit and then hitchhiked to the periplasm via the Tat pathway [18]. This is similar to the mechanism proposed for the periplasmic nitrate reductase [38]. However, unlike NapC, the γ-subunit of (per)chlorate reductase is not a membrane-bound protein. As a result, a mediator is required for electron transfer to the (per)chlorate reductase. This mediator is a 6 kDa periplasmic cytochrome c-containing peptide, which has recently been described for I. dechloratans. Analogues of this periplasmic c cytochrome have also been found in the genome of D. aromatica [39].

Figure 1 Multiple sequence alignment of the α-subunits of chlorate reductase (ClrA) of I. dechloratans (Id), (per)chlorate reductase (PcrA) of Dechlorosoma KJ (KJ), Dechlorosoma PCC (PC) and D. agitata (Da), and of nitrate reductase (NarG) of E. coli (Ec) Pseudomonas fluorescens (Pf) and Bacillus subtilis (Bs)

Protein sequences were obtained from the GenBank® database [49]. GenBank® accession numbers are as follows: ClrA Id, P60068; PcrA KJ, ACB69917; PcrA PC, ABS59782; PcrA Da, AAO49008; NarG Ec, CAQ32609; NarG Pf, AAG34373; and NarG Bs, P42175. The conserved aspartate residue which co-ordinates Mo is shaded and marked with an asterisk.

Phylogenetic analyses showed that the (per)chlorate reductases of two Dechloromonas strains, D. agitata and D. aromatica, formed a separate monophyletic group from the chlorate reductase of I. dechloratans. The analyses indicated that (per)chlorate reductases are more closely related to nitrate reductases than to chlorate reductases [17]. Our phylogenetic analysis supports this (Figure 2). This observation can also be deduced from the physiological properties of the known (per)chlorate reducers. Azospira oryzae strain GR1 and Dechloromonas hortensis reduce perchlorate as well as nitrate efficiently, whereas some of the chlorate-reducers such as Pseudomonas sp. PDA and P. chloritidismutans cannot efficiently reduce nitrate [10,12,40,41]. Our phylogenetic studies identified that a putative nitrate reductase subunit, the NarG of Sagittula stellata, shows high similarity to the ClrA of I. dechloratans (Figure 2). Since S. stellata cannot reduce nitrate [42], this putative NarG might be ClrA instead.

Figure 2 Unrooted neighbour-joining tree indicating the evolutionary distances of NarG, NapA, PcrA and ClrA, the α-subunits of respiratory nitrate reductase, (per)chlorate reductase and chlorate reductase respectively

Protein sequences were obtained from the GenBank® database [49]. GenBank® accession numbers are indicated. The tree was assembled using ClustalX 1.83 [50], visualized using TreeView 1.6.6 software [developed by R.D.M. Page at the University of Glasgow (], and any make-up was performed using CorelDraw version 12.0. This unrooted tree was bootstrapped with the number of bootstrap trials set to 1000. B. subtilis, Bacillus subtilis; H. influenzae, Haemophilus influenzae; M. multacida, Mitsuokella multacida; P. aeruginosa, Pseudomonas aeruginosa; P. aerophilum, Pyrobaculum aerophilum; P. arsenaticum, Pyrobaculum arsenaticum; P. calidifontis, Pyrobaculum calidifontis; P. fluorescens, Pseudomonas fluorescens; P. stuttzeri, Pseudomonas stutzeri; R. equi, Rhodococcus equi; R. eutropha, Ralstonia eutropha; R. sphaeroides, Rhodobacter sphaeroides; S. coelicolor, Streptomyces coelicolor; V. atypica, Veillonella atypica; V. parvula, Veillonella parvula.

Putative (per)chlorate reduction by nitrate reductases based on sequence similarities

In denitrification, after nitrate reduction to nitrite, dinitrogen is formed by the successive action of nitrite reductase, nitric oxide reductase and nitrous oxide reductase. However, in the (per)chlorate reductase pathway, instead of three enzymes, only a single enzyme, chlorite dismutase, converts chlorite into chloride [25].

A large number of genes similar to chlorite dismutase were found in sequenced genomes, indicating their putative (per)chlorate-reduction potential [43]. We have searched for the presence of putative nitrate reductases of the Nar-type in bacteria containing putative chlorite dismutase. Proteobacteria that are known chlorate reducers were ignored. Bacteria that contain a putative chlorite dismutase and the catalytic subunit of a putative respiratory nitrate reductase are shown in Supplementary Table S1 at As in the study by Maixner et al. [43], the distribution of chlorite dismutase-like sequences was widespread, although the majority of micro-organisms containing chlorite dismutase-like sequences in putative respiratory nitrate reductase-encoding genes were Bacillus and Staphylococcus species (Supplementary Table S1). The frequent distribution of chlorite dismutase-like genes of putative (per)chlorate-reducing micro-organisms indicates the ubiquity of (per)chlorate reduction among micro-organisms. From an environmental point of view, Mycobacterium KMS, which is a known aerobic pyrene-degrading micro-organism [44] and Geobacillus thermodenitrificans NG80-2, a known aerobic long-chain alkane degrader [45], are interesting bacteria (Supplementary Table S1).


Prokaryotic nitrate and chlorate reduction pathways have many similarities and some variations. The nitrate reduction pathway has been studied in more detail than the (per)chlorate reduction pathway. In the present review, we have shown that there are similarities in the enzyme structures and sequences of (per)chlorate reductase and dissimilatory nitrate reductase. Unfortunately, a crystal structure of a (per)chlorate reductase is not yet available. Many supplementary genes in the (per)chlorate reduction pathway are still missing, e.g. the regulatory genes such as Fnr, ResD, ResE and Nar XL for the nitrate reduction pathway. This shows that our understanding of (per)chlorate reduction is far from complete. Further studies of nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria are needed. Analysis of gene sequences encoding chlorite dismutase have indicated that the capacity to reduce (per)chlorate may be widespread among micro-organisms. This is not surprising, since (per)chlorate is naturally occurring. Recent evidence of another pathway of nitrate reduction which has similarities to the (per)chlorate reduction pathway has been presented. The proposed pathway involves the as yet hypothetical ‘nitric oxide dismutase’ enzyme, which, like chlorite dismutase, releases molecular oxygen [13]. The potential of nitrate and chlorate to enhance biodegradation of recalcitrant pollutants in anaerobic soils needs further study, as the use of oxygenase-dependent pathways under seemingly anoxic conditions offers attractive possibilities.


This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO) [project number 08053].


We thank the members of the users committee of STW project 08053 for discussion and advice.


  • Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

Abbreviations: MGD, molybdopterin guanine dinucleotide; Moco, molybdenum cofactor; Tat, twin-arginine translocation


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