The canonical structural motif for co-ordination of non-haem ferrous iron in metal-dependent oxygenases is a facial triad of two histidine residues and one aspartate or glutamate residue. This so-called 2-His-1-carboxylate metallocentre is often accommodated in a double-stranded β-helix fold with the iron-co-ordinating residues located in the rigid core structure of the protein. At the sequence level, the metal ligands are arranged in a HXD/E…H motif (where the distance between the conserved histidine residues is variable). Interestingly, cysteine dioxygenase, among a growing number of other iron(II) oxygenases, has the carboxylate residue replaced by another histidine. In the present review, we compare the properties of 3-His and 2-His-1-carboxylate sites based on current evidence from high-resolution crystal structures, spectroscopic characterization of the metal centres and results from mutagenesis studies. Although the overall conformation of the two metal sites is quite similar, the carboxylate residue seems to accommodate a slightly closer co-ordination distance than the counterpart histidine. The ability of the 2-His-1-carboxylate site to fit a site-directed substitution by an alternatively co-ordinating or non-co-ordinating residue with retention of metal-binding capacity and catalytic function varies among different enzymes. However, replacement by histidine disrupted the activity in the three iron(II) oxygenases examined so far.
Non-haem iron-dependent enzymes constitute a functionally diverse group of proteins that catalyse a remarkably wide spectrum of chemical reactions. Many of them use O2 as co-substrate and perform oxygenase and oxidase-type catalytic transformations, including hydroxylations, ring expansions, oxidative cyclizations, desaturations, oxidations and epoxidations. The iron centre plays a crucial role in enzymatic catalysis as it is normally used for co-ordination and catalytic activation of the substrate and O2. The non-haem iron enzymes can be distinguished according to whether they employ α-oxoglutarate (α-ketoglutarate) as an essential co-substrate. The structure–function relationships and mechanistic properties of the α-oxoglutarate-dependent group of enzymes have received much attention in the last few years [1–7]. In spite of the different reactions promoted, the catalytic pathways of α-oxoglutarate-dependent enzymes are oftentimes united by the occurrence of a common high-valent iron(IV)-oxo intermediate that fulfils a central role during substrate oxygenation [8,9].
The 2-His-1-carboxylate facial triad
A key feature of structure and function in most of the currently known non-haem iron enzymes is a conserved active-site conformation in which a triad of residues, two histidine residues and one aspartate or glutamate residue, utilize their side chains to co-ordinate the catalytic metal . Although similarity in amino acid sequence is often hardly significant among these enzymes, the so-called 2-His-1-carboxylate motif of non-haem iron is widely conserved at the level of the three-dimensional protein structure and spans different fold families. There has been much recent progress in X-ray structure determination of non-haem iron enzymes and also in the detailed biochemical and biophysical characterization of the corresponding metallocentres, either in natural proteins or in biomimetic synthetic complexes [1,11]. The combined evidence from these studies portrays a diverse landscape of catalytic mechanisms that is accessible to the 2-His-1-carboxylate centre of non-haem iron. The well-developed structural basis for these enzymes promotes the comparative analysis of the role of the protein environment of the catalytic metal on the type of reaction catalysed as well as on selectivity and efficiency of the enzymatic transformation. It also aids in advancing the currently limited understanding of how selectivity for the binding of iron is achieved by the 2-His-1-carboxylate facial triad.
Alternative structural motifs for the co-ordination of iron
It has recently become clear that alternative structural motifs for binding of iron(II) have evolved in natural proteins alongside the canonical 2-His-1-carboxylate metallocentre . CDO (cysteine dioxygenase) (Figure 1) , GDO (gentisate 1,2-dioxygenase)  and Dke1 (diketone-cleaving enzyme 1)  use three histidine residues as ligands to the iron cofactor. QDO (quercetin dioxygenase)  and ARD (acireductone dioxygenase)  employ essentially the same 3-His structural motif for metal co-ordination, but, in contrast with CDO, GDO and Dke1, utilize an additional glutamate residue to ligate the iron in their respective resting states. Also in pirin, a nuclear protein shown previously to possess quercetinase activity , ferrous iron is co-ordinated by a 3-His-1-Glu motif . In the halogenase SyrB2, the carboxylate residue of the 2-His-1-carboxylate facial triad is replaced by an alanine residue. The X-ray structure of SyrB2 shows that the space around the catalytic iron thus vacated is occupied by a halide ion. It seems that binding of the chloride substrate of the enzymatic reaction partly restores the character, structurally and charge-wise, of the canonical 2-His-1-carboxylate centre .
Crystal structure of mouse CDO (PDB code 2B5H)
The DSBH (double-stranded β-helix) fold
Irrespective of the residues utilized to create the primary co-ordination sphere for iron, the overall geometry of the metal centre is remarkably conserved among non-haem iron enzymes, as shown in Figure 2 where the 3-His site in CDO  and the 2-His-1-Asp site of TauD (taurine/α-oxoglutarate-dependent dioxygenase)  are compared. Interestingly, structural similarity among the 3-His group of enzymes (CDO, GDO, Dke1, QDO, ARD and pirin) is likewise detected at the level of the overall protein fold which, in each case, comprises a DSBH core (Figure 1). Note that the DSBH fold has also evolved various 2-His-1-carboxylate centres . The ligands to the iron cofactor (3-His or 2-His-1-Asp/Glu) are contributed from a conserved HXD/E/H…H motif where the distance between the first and second conserved histidine residue (in bold) is variable, being between approx. 42 and 193 residues. Analysis of the available X-ray structures for annotated mononuclear non-haem iron oxygenases (excluding iron–sulfur cluster proteins) reveals that the DSBH fold is predominantly (21 out of 30 proteins) used. We note that the nomenclature employed in the literature is somewhat ambiguous because proteins assigned to the structural superfamilies of cupins  and JmjC transcription factors  display many features typical of the DSBH fold. These common characteristics include a pair of four-stranded antiparallel β-sheets constituting up to eight β-strands which in turn form the typical β-sandwich structure. The positions of the metal-co-ordinating residues are strictly conserved on the inside of the rigid core of the overall protein structure. Figure 1 shows the geometry of the iron-binding site of rat CDO (PDB code 2B5H) with the typical DSBH fold. ARD presents a special case because one of the histidine ligands is flexibly located in the middle on a loop between two strands of the β-sandwich structure .
Superposition of the metal centres of CDO (PDB code 2B5H) in white and TauD (PDB code 1OS7) in yellow
We compared the 2-His-1-carboxylate and 3-His motifs of non-haem iron in DSBH proteins with respect to their structural and functional properties. Results of site-directed mutagenesis studies designed to examine the role of primary co-ordination sphere residues in 2-His-1-carboxylate enzymes are also reviewed. Among the various point mutants reported in the literature, there are few where, unintentionally, the original 2-His-1-carboxylate centre for non-haem iron was converted at the sequence level into one that now contained three histidine residues. Special focus is placed on the functional properties of such mutants.
3-His iron centres, and enzymatic reactions catalysed by them
Metallocentres comprising three histidine ligands have been primarily described for enzymes that bind zinc in their active site. The zinc-dependent carbonic anhydrase is a well-studied 3-His metalloenzyme that serves as a representative example . Among the group of non-haem iron enzymes, however, the 3-His metallocentre has only recently drawn more widespread attention, especially after it was shown that CDO co-ordinates its iron cofactor via the side chains of three histidine residues. CDO catalyses the O2-dependent conversion of cysteine into cysteine sulfinic acid  and fulfils a physiological function in cysteine homoeostasis of mammalian tissues. The enzyme is considered to be a potential drug target in therapies addressing the imbalance in the metabolism of cysteine which is a known accompaniment of Parkinson's or Alzheimer's disease . The crystal structures of human , rat  and mouse  CDO were solved recently and all show the mononuclear non-haem iron(II) centre to consist of three histidine residues.
Gentisic acid acts as important intermediate in bacterial metabolism of aromatic compounds. It is converted into maleylpyruvate by GDO through an oxygen-dependent reaction in which the aromatic ring of the substrate is opened. The structure of GDO from Escherichia coli revealed a bicupin fold that features two DSBH regions (PDB code 2D40). The active-site Fe(II) is ligated by a 3-His triad. The remaining co-ordination sites of ferrous iron are occupied by water molecules when the enzyme is in the resting state . Recently, another crystal structure for GDO from Silicibacter pomeroyi (PDB code 3BU7) became available, and this enzyme closely resembles the GDO from E. coli .
The crystal structure of Dke1 from Acinetobacter johnsonii was determined for an inactive zinc-bound variant of the native enzyme that requires iron(II) for activity (PDB code 3BAL). The enzyme is a dioxygenase that cleaves carbon–carbon bonds in β-diketone substrates via the incorporation of one atom of molecular oxygen into each site of bond fission . Acetylacetone (2,4-pentanedione) is therefore converted into methylglyoxal and acetate. The structure reveals co-ordination of the catalytically incompetent zinc via three histidine residues and the conformation of the metallocentre of Dke1 is highly similar to those seen in CDOs.
ARD co-ordinates its active-site metal (Fe2+ or Ni2+) by three histidine residues plus an additional glutamate residue provided from the enzyme . This glutamate (Glu-102) is partly conserved at the sequence level and corresponds to Glu-69 of Dke1. However, the structure of Dke1 shows that Glu-69 is not co-ordinating and points away from the active site. Site-directed mutagenesis was used to investigate the function of Glu-69 in Dke1. A variant enzyme harbouring a replacement of Glu-69 by glutamine displayed similar properties to those of the wild-type enzyme . This therefore suggested that the conserved glutamate residue is not important for enzyme function or that it has a different role in ARD compared with Dke1. Depending on the metal bound at the 3-His-1-Glu centre, ARD catalyses two different reactions. ARD with ferrous iron bound promotes the cleavage of 1,2-dihydroxy-5-methylthiopent-1-en-3-one to a precursor of methionine and formate in the methionine salvage pathway in Klebsiella pneumoniae. The Ni2+-bound variant catalyses an off-pathway reaction, leading to products methylthiopropionate, CO and formate thus preventing the recycling to methionine [32,33].
QDOs constitute a group of dioxygenases that display remarkable variability and promiscuity with respect to the metal used in catalysis. Cu2+, Mn2+, Ni2+, Co2+ and Fe3+ were reported to replace Fe2+ as the active-site metal in the conversion of quercetin by QDO from Bacillus subtilis . The ligand environment for the catalytic metal in QDO in the resting state is the same as in ARD. However, since, in both ARD and QDO, the glutamate residue is proposed to move away from its metal-co-ordinating position during the course of catalytic action, the metallocentre in its active form appears to be of the 3-His type and is therefore considered in our comparisons. The transformation catalysed by QDO is the O2-dependent cleavage of the O-heteroaromatic ring of flavonols such as quercetin [34,35].
The in vivo function of pirin has not been clearly resolved yet, but it was suggested that it serves a role as transcription cofactor. The enzyme is widespread in mammals (including humans), plants, fungi and prokaryotes . There are crystal structures for human pirin (PDB code 1J1L) and a pirin homologue from E. coli (PDB code 1TQ5). The human protein showed a 3-His-1-Glu metal-binding site . The structure of the E. coli protein was solved in the presence of Cd2+. The cadmium was co-ordinated only by two histidine residues of the potential 3-His metal site . Interestingly, quercetin was reported to act as substrate for pirins in a similar way as it does for QDO .
Structural comparison of 3-His and 2-His-1-carboxylate centres of non-haem iron
Location of the metal centre
The residues ligating the iron cofactor in CDO, TauD and likewise the other 3-His enzymes discussed herein are located in the very rigid core of the protein fold and can normally be found on the end of strand 2, with the distal histidine residue being contributed by strand 7 . In CDO, the active site is buried deep inside the protein core, approx. 8 Å (1 Å=0.1 nm) away from the surface and embedded in a solvent-filled environment . Both the core structure and the active-site residues of TauD align very well with CDO, the main difference being the presence of few additional α-helices and β-sheets towards the surface of the protein that are appended in TauD.
Conformation of the metal centre
3-His and 2-His-1-carboxylate centres of non-haem iron show a remarkably high degree of structural similarity. Figure 2 depicts a structural superimposition of the active sites of CDO (PDB code 2B5H) and TauD (PDB code 1OS7). The spatial arrangement of metal ligands is very similar in both enzymes despite the non-conservative exchange of the uncharged side chain of the histidine residue in CDO by the negatively charged side chain of the aspartate residue in TauD. The structural overlay also indicates that Asp-101 in TauD and His-88 in CDO adopt the same position in the protein, and both enzymes feature identical iron-binding geometries. However, a change in the orientation of the distal histidine usually located on strand 7 in DSBH proteins can be observed.
Iron is preferably co-ordinated in an octahedral manner with a facial arrangement of the metal ligands. Therefore three cis-sites are left open on the iron cofactor for the binding of substrate, co-substrate and oxygen. Crystal structures and evidence from XAS (X-ray absorption spectroscopy) independently confirmed an average co-ordination distance of 2.1 Å for the histidine side chains that ligate iron(II) in CDO [21,37]. The metal–ligand distances in TauD are longer than the corresponding co-ordination distances in CDO (Table 1). We therefore analysed reported crystallographic and XAS data for non-haem metal centres in DSBH-fold oxygenases (Table 1) and determined averaged iron–ligand distances of 2.14±0.10 and 2.19±0.12 Å for carboxylate O and histidine N donor groups respectively. Precision of the overall analysis is, however, limited by the relatively broad range of experimental metal–ligand distances of between 2.05 and 2.5 Å found in the database. However, our findings are in reasonable agreement with the work of Harding  who described average distances of 2.04 and 2.16 Å for aspartate/glutamate carboxylate O and histidine imidazole N ligands to ferrous iron. It would therefore seem that the co-ordination distance of carboxylate groups in non-haem Fe2+ centres is significantly shorter than that of imidazole groups. In addition to the difference in overall net charge of 3-His iron sites compared with the 2-His-1-carboxylate counterparts, the aspect of metal–ligand distance could be another feature of structural and perhaps functional distinction among the two metal centres.
|Enzyme||Ligand||Metal–ligand distances (Å)||PDB code||Reference|
|TauD||His-99 (NE2)||2.48||1OS7 chain c|||
|Dke1||His-62 (NE2)||2.24||3BAL† chain a|||
|Enzyme||Ligand||Metal–ligand distances (Å)||PDB code||Reference|
|TauD||His-99 (NE2)||2.48||1OS7 chain c|||
|Dke1||His-62 (NE2)||2.24||3BAL† chain a|||
Determined by XAS.
Active-site metal is zinc.
Mutational analysis of protein centres of non-haem iron
The primary co-ordination sphere of ferrous iron in various enzymes of the 2-His-1-carboxylate type has been examined by site-directed mutagenesis. Note that comprehensive coverage of previous works was not an intention of the present paper, and the reader is referred to a detailed review . From evidence obtained at low- (e.g. [40,41]) and molecular- (e.g. ) level resolution, it can be assumed that the overall protein structure has, in general, remained unaffected in the enzyme variants prepared. Biochemical studies conducted with a number of DSBH enzymes suggest that, upon point mutation of one iron-co-ordinating residue by a non-co-ordinating or alternatively co-ordinating amino acid, the metal centre is usually not globally disrupted and often retains some of the original affinity for ferrous iron [39,42–45]. Only in few cases, however, the mutated metal enzymes show the ability to promote efficiently the reactions catalysed by the wild-type form [39,41,46,47]. Crystal structures of mutants of FIH [factor inhibiting HIF (hypoxia-inducible factor)], a hydroxylase involved in the regulation of hypoxic response in humans in which Asp-201 had been replaced by alanine or glycine, confirmed an otherwise intact metal centre and revealed that only minor structural rearrangements had occurred around the substrate-binding site as result of the site-specific substitutions .
TauD , IPNS (isopenicillin N synthase)  and ethylene-forming enzyme ACCO (1-aminocyclopropane-1-carboxylate oxidase)  all co-ordinate ferrous iron by a HXD…H facial triad. Replacement of the aspartate residue by histidine formally converts the original 2-His-1-Asp centre into one where iron(II) now has three histidine ligands. The relevant aspartate→histidine mutants of the three enzymes were completely inactive in the O2-dependent transformation catalysed by the respective wild-type oxygenase [39,50,51]. It is interesting, however, that IPNS mutant D214H was still able to bind iron and, in isolated preparations, contained the same amount of metal as the wild-type enzyme (0.65 equivalents) .
The influence of the length of the carboxylate side chain in 2-His-1-carboxylate centres was examined by replacing aspartate by glutamate. The activity of the relevant aspartate→glutamate mutant of IPNS was only 1%  and that of tomato ACCO below 0.2%  compared with the level of respective wild-type enzyme. The pronounced loss of activity caused by the mutation can probably be attributed to structural rigidity in and around the active sites of IPNS and ACCO which seems to prevent the accommodation of the longer side chain of glutamate. TauD and the α-oxoglutarate-dependent human prolyl 4-hydroxylase can cope better than IPNS and ACCO with the extra steric bulk resulting from the substitution of glutamate for aspartate, their aspartate→glutamate mutants retaining 22% and 15% of wild-type activity respectively [39,46].
Perhaps surprisingly, considering the absence of detectable activity in the 3-His variant of TauD, this enzyme could accommodate a site-directed substitution of its distal His-255 by glutamate and glutamine with retention of 33% and 81% of wild-type activity respectively . In aspartyl (asparagyl) β-hydroxylase, mutated forms H675E and H675D were 12 and 20% as active as the wild-type enzyme respectively . However, analogous changes at the metal-binding site of tomato ACCO brought about a far more substantial disruption (>99.8%) of the original activity . It is therefore interesting that, in spite of the structural similarity of the primary co-ordination sphere of ferrous iron in DSBH enzymes of the 2-His-1-carboxylate type, the consequences of site-directed replacements in the metal centres on iron binding and catalytic function vary widely and apparently with no clear pattern.
Another interesting feature of 2-His-1-carboxylate centres is that the position of the negatively charged residue in the facial triad seems to play an important role in function, at least in certain enzymes. Swapping the positions of histidine and aspartate resulted in inactive doubly mutated IPNS (H212D/D214H and D214H/H268D ) and tomato ACCO (H177D/D179H ). The iron content of IPNS D214H/H268D was also reduced to 0.09 equivalents .
Considering the marked disruption of iron binding in various 2-His-1-carboxylate enzymes upon replacement of one metal-co-ordinating residue by the non-co-ordinating alanine , it is quite interesting to examine the halogenase SyrB2 in which co-ordination of ferrous iron is achieved by using only two protein-derived ligands, namely two histidine residues. The third position of the otherwise highly conserved metal centre is taken by alanine, and the space vacated between iron and the protein allows binding of a halide ion, which serves as co-substrate in the enzymatic reaction . The occurrence of SyrB2 raises the question of the minimal protein centre of non-haem iron(II). Definitive answers are currently elusive. However, mutagenesis work has shown that some enzymes can tolerate removal of a co-ordinating residue at a certain position with partial retention of functionality. The H99A mutant of TauD, for example, still showed about half the original enzyme activity . Very recently, the truncated 2-His-(1-Ala/Leu) version of the original 2-His-1-Asp centre for non-haem iron(II) in FIH was found to be functional with respect to iron binding and catalysis to the oxidative decarboxylation of α-oxoglutarate . In human PAHX (phytanoyl-CoA hydroxylase) and CAS (clavaminic acid synthase), the distal histidine residue of the facial triad was mutated to alanine and leucine, the remaining activities being 7.% and 3% of the wild-type level respectively [43,44].
ARD is only one of two 3-His enzymes in which the metal centre has been subject to mutational analysis. Individual site-directed replacement by alanine of the residues co-ordinating the iron(II) in the resting enzyme led to variants that were either poorly soluble under conditions of recombinant protein production (E102A and H140A) or showed very disrupted function (H96A and H98A), so that the measured activity was not separable from the background level .
For GDO (from S. pomeroyi), each of the two metal-binding sites of the bicupin structure was probed by site-directed mutagenesis. The doubly mutated enzymes H119A/H121A and H290A/H292A were created to eliminate two co-ordinating residues in the respective metal site at a time. Although the variant on the N-terminal domain was insoluble, the enzyme harbouring mutations in the C-domain was expressed functionally and characterized by CD, crystal structure analysis and activity assays. Although the overall structure of the doubly mutated enzyme was very similar to that of wild-type GDO, there was hardly any activity detectable for the GDO variant, even when using high protein concentrations .
Comparison of 2-His-1-carboxylate and 3-His centres of non-haem iron(II) reveals that, on top of a striking overall structural similarity in DSBH proteins (Figure 1), there are also small differences in ligand orientation (Figure 2) and in the preferred metal-ligand distances for the conserved histidine residues (Table 1). The functional significance of these differences is not known and should be explored in future work, focusing on the interconversion of 2-His-1-carboxylate and 3-His sites in natural non-haem iron enzymes. The currently available evidence obtained from study of a quite limited number of enzymes indicates that the substitution of histidine for aspartate or glutamate is disruptive with respect to the naturally occurring type of oxygenase activity. Therefore more attention should be paid to the role in catalysis of the secondary co-ordination sphere of the iron(II) cofactor. Fine-tuning of reactivity is often achieved by residues that interact directly or through a hydrogen-bond network with first sphere ligands. However, although many details are still lacking, the current picture portrays the 3-His metal centre as a distinct motif of structure and function in non-haem iron(II)-dependent enzymes.
Transition Metals in Biochemistry: A joint Biochemical Society meeting with the Inorganic Biochemistry Discussion Group to honour Professor Andrew Thomson FRS, held at University of East Anglia, Norwich, U.K., 24–26 June 2008. Organized and Edited by Steve Chapman (Edinburgh, U.K.), David Richardson (University of East Anglia, U.K.) and Nick Watmough (University of East Anglia, U.K.).
factor inhibiting HIF (hypoxia-inducible factor)
isopenicillin N synthase
X-ray absorption spectroscopy