A number of biochemically distinct systems have been characterized for the microbial reduction of the oxyanions, selenate (SeO42−) and nitrate (NO3−). Two classes of molybdenum-dependent nitrate reductase catalyse the respiratory-linked reduction of nitrate (NO3−) to nitrite (NO2−). The main respiratory nitrate reductase (NAR) is membrane-anchored, with its active site facing the cytoplasmic compartment. The other enzyme (NAP) is water-soluble and located in the periplasm. In recent years, our understanding of each of these enzyme systems has increased significantly. The crystal structures of both NAR and NAP have now been solved and they provide new insight into the structure, function and evolution of these respiratory complexes. In contrast, our understanding of microbial selenate (SeO42−) reduction and respiration is at an early stage; however, similarities to the nitrate reductase systems are emerging. This review will consider some of the common themes and variations between the different classes of nitrate and selenate reductases.
- nitrate reductase
- selenate reductase
Inorganic nitrogen in the form of nitrate (NO3−) is widely and readily available for plants and microorganisms. Selenium (Se) in contrast, is a naturally occurring trace element in very low abundance. Under aerobic conditions Se is present in several redox forms, including elemental Se, although it exists predominantly as the high-valence toxic and soluble oxyanions, selenate (SeO42−) and selenite (SeO32−). Microbes, which can reduce these selenium oxyanions, are not restricted to any particular group/subgroup of prokaryotes and examples are found throughout the bacterial domain. However, for some species, including Thauera selenatis, Sulfurospirillum barnesii, Enterobacter cloacae and Aeromonas hydrophila, the reduction of selenate is linked to the respiratory electron transfer chain.
The reduction of selenate has often been associated with the process of denitrification, with all selenate reducers also capable of reducing nitrate. On the basis of these observations, selenate reduction has often been suggested to be an additional side reaction of the respiratory nitrate reductases [1,2] and, indeed, the selenate reductase activity of both the membrane-bound (NAR) and periplasmic (NAP) nitrate reductases from Paracoccus denitrificans and Paracoccus pantotrophus has been demonstrated (Table 1). It is evident however that nitrate reductases are poor reducers of selenate [1–3] and may not contribute significantly to global selenate reduction, particularly in areas enriched with both selenate and nitrate. Consequently, novel enzyme systems that catalyse the reduction of selenate selectively have been sought and recent biochemical and genetic studies suggest that, as for nitrate, distinct classes of enzymes that catalyse the selective reduction of selenate do exist. In this short review, we consider the recent advances in the characterization of classes of nitrate reductase (NAP and NAR) and compare their similarities with the emerging selenate reductases.
Periplasmic nitrate reductase
The periplasmic nitrate reductase (NAP) is linked to quinol oxidation in the respiratory chain, but has been shown to have a range of functions in different bacteria, including nitrate respiration under nitrate-limited conditions and in the disposal of excess reducing equivalents during aerobic growth . Despite these differences in primary function, the NAP family of enzymes possess a similar structure, with NAP from P. pantotrophus and Escherichia coli comprising three subunits. A membrane-anchored tetra-haem c-type cytochrome component NapC mediates electron flow between quinol and the catalytic NapAB complex [5,6]. NapB is a soluble 16 kDa subunit of c-type cytochrome , which transfers the electrons to the 80–90 kDa catalytic subunit NapA. NapA has an N-terminal [4Fe-4S] cluster co-ordinated by four cysteine ligands  and also contains the active-site molybdenum centre, comprised of a bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor [3,6–8]. Mo in NAP is co-ordinated to the protein through a thiolate ligand provided by a cysteine residue [6,8]. The location of the active site in the periplasmic compartment means that the two protons pumped across the membrane by NapC are subsequently consumed, as NapA catalyses the reduction of nitrate to nitrite with the concomitant production of H2O . As a consequence, NAP does not directly contribute to the generation of the proton motive force.
Membrane-bound nitrate reductase
The membrane-bound nitrate reductase (NAR) is a heterotrimer with subunits NarG (∼140 kDa), NarH (58 kDa) and NarI (20 kDa) forming a flower-shaped structure with dimensions of 90 Å×128 Å×70 Å (1 Å=0.1 nm) . NarI anchors the other subunits to the cytoplasmic membrane mainly through hydrophobic interactions. The haem groups (two b-type cytochromes) in NarI receive electrons from the quinol pool and these are then transferred, through a series of iron–sulphur clusters (in NarH), to the catalytic subunit NarG. The recent crystal structures of NAR from E. coli have shown that NarG contains an unusual [4Fe-4S] cluster, previously undetected by EPR spectroscopy, co-ordinated by one histidine and three cysteine ligands [9,10]. Furthermore, the Mo centre in the bis-MGD cofactor is co-ordinated to the peptide through a previously uncharacterized aspartic residue (Asp222) [9,10]. For the first time, the structural information has shed light on what may control substrate specificity. The presence of an asparagine residue (Asn52), 3.9 Å away from the Mo, may provide a suitable and open site for substrate binding.
Periplasmic selenate reductase
The selenate reductase from T. selenatis is a periplasmic trimeric enzyme with an apparent molecular mass of approx. 180 kDa. The three subunits consist of SerA (96 kDa), SerB (40 kDa) and SerC (23 kDa) (Figure 1). The SerA subunit has an N-terminal cysteine-rich motif, probably co-ordinating a [4Fe-4S] cluster as seen in NapA, and also contains the Mo active site. The SerB subunit also has a number of cysteine-rich motifs, which again suggest the presence of iron–sulphur clusters. The SerC subunit contains b-type cytochrome as shown by absorption spectroscopy . This is in contrast with NapB that contains two c-type cytochromes. DNA sequence analysis from T. selenatis has identified the presence of a fourth component (SerD) that may function as a specific chaperone assembly protein, analogous to NapD which might be involved in cofactor insertion into SerA . A membrane-bound component analogous to NapC or NarI has not yet been identified, so the process by which SerABC receives electrons from the quinol pool remains to be established. The selenate reductase shows surprising substrate specificity and does not reduce nitrate, chlorate or sulphate . Amino acid sequence alignment of the periplasmic selenate reductase (SerA) with the periplasmic (NapA) and membrane-bound (NarG) nitrate reductases has shown that SerA is more closely related to NarG than NapA, despite its periplasmic location. The highly conserved aspartic residue (see alignment below) shown to co-ordinate the Mo in NAR is also present in SerA, placing SerA as a member of the type II molybdo-enzymes .
Interestingly, however, recent analysis of the selenate reductase by EXAFS has suggested that the active site resembles that of arsenite oxidase and does not have an amino acid side-chain ligand to the Mo . The asparagine residue (Asn52) conserved in the active site of NarG is replaced with glycine (Gly76) in SerA and may be important in controlling substrate specificity.
Membrane-bound selenate reductase
A membrane-bound selenate reductase has been identified in E. cloacae SLD1a-1 and S. barnesii but has been studied to a far lesser extent than its nitrate reductase cousin. The enzyme from S. barnesii has been purified and is a heterotetramer with subunits of 82, 53, 34 and 21 kDa . To date the analysis of the redox centres of the purified enzyme has not been reported, but a b-type cytochrome spectrum has been detected in the membrane fraction from selenate grown cells , suggesting the presence of a subunit analogous to NarI. The membrane fractions isolated from S. barnesii exhibited greatest activity towards selenate, but also reduced the substrates' nitrate, thiosulphate and fumarate, even though components of these pathways were not readily detected . These results suggest that this membrane-bound selenate reductase has much broader substrate specificity than the periplasmic selenate reductase from T. selenatis.
Recently we have undertaken a study of selenate reduction by E. cloacae SLD1a-1 and have demonstrated conclusively that two distinct membrane-bound reductases are expressed for the reduction of nitrate and selenate . The nitrate reductase is typical of the NAR family in its composition (NarG∼150 kDa and NarH∼60 kDa). However, unlike P. denitrificans, E. cloacae SLD1a-1 can grow using nitrate respiration in the presence of high concentrations (10 mM) of selenate, suggesting a functional role for a separate selenate reductase. The selenate reductase is also membrane-bound and has a complex mass of approx. 600 kDa as determined by gel filtration. The enzyme is inhibited by tungstate and activated by molybdate, suggesting that it is a molybdo-enzyme. It is expressed predominantly under aerobic conditions (Table 1)  and, unlike the S. barnesii enzyme, this enzyme appears specific for selenate and displays no activity towards nitrate . We have further demonstrated that the selenate reductase of E. cloacae SLD1a-1 is orientated such that its active site faces the periplasmic compartment  (Figure 1), opposite to the NarGH components of the membrane-bound nitrate reductase. This orientation may well suit its primary function of detoxification and protect the cell against the toxic effects of both selenate and the reduction intermediate selenite .
Our understanding of microbial selenate reduction is still at a very early stage; however, similarities to the respiratory nitrate reductase systems are apparent. It is hoped that through the recent structural studies of the nitrate reductases, together with biochemical and genetic studies of the selenate reductases, key factors that govern substrate specificity of these and other related molybdo-enzymes can be elucidated.
This work was supported by BBSRC research grants 13/P17219 and BBS/B/10110. E.J.D. and J.T.L. are recipients of BBSRC PhD studentships.
The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).
Abbreviations: bis-MGD, bis-molybdopterin guanine dinucleotide
- © 2005 The Biochemical Society