Metal-binding Ligands Research Articles

Triostin A derived hybrid for simultaneous DNA binding and metal coordination.

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7]triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-010-0764-3) contains supplementary material, which is available to authorized users.

Modulation of MnSOD protein in response to different experimental stimulation in Hyphantria cunea

A manganese superoxide dismutase (MnSOD) gene was cloned from the fall webworm, Hyphantria cunea. MnSOD cDNAs encode precursor proteins of 215 amino acid residues. H. cunea MnSOD possesses the metal binding ligands of 3 histidines and 1 aspartic acid common to MnSODs. The deduced amino acid sequences of the H. cunea MnSOD cDNA showed 76% identity to Bombyx mori MnSOD and 56-62% identity to MnSOD sequences from other species. MnSOD and copper/zinc superoxide dismutase (Cu/ZnSOD) is expressed in all tissues of H. cunea. MnSOD expression changed at a trace-level in infected larvae, while Cu/ZnSOD expression strongly changed against Gram-positive and Gram-negative bacteria, and fungi. Environmental stresses such as different artificial photoperiods (24L:0D), ultraviolet irradiation (312 nm), and starvation condition increased Cu/ZnSOD expression, MnSOD expression, on the other hand, was increased by starvation. Moreover, MnSOD and Cu/ZnSOD expression showed no significant change in the 0L:24D condition. MnSOD and Cu/ZnSOD expression in H. cunea also significantly increased at high (37°C) and low (4°C) temperature. Oxidative stress induced by 10% H(2)O(2) reduced the expression levels of MnSOD and Cu/ZnSOD. However, paraquat-induced oxidative stress reduced MnSOD expression but increased Cu/ZnSOD expression. These results suggest that Cu/ZnSOD may play a larger role than MnSOD as a superoxide anion scavenger against oxidative stress in H. cunea.

Polymer-based fluorescence sensor incorporating triazole moieties for Hg2+ detection via click reaction

The polymer could be obtained by the polymerization of 1,4-dibutoxy-2,5-diethynylbenzene (M-1) with 1,4-diazidobenzene (M-2)via click reaction. The polymer show blue fluorescence. The responsive optical properties of the polymer on various transition metal ions were investigated by fluorescence spectra. Compared with other cations, such as Co2+, Ni2+, Ag+, Cd2+, Cu2+ and Zn2+, Hg2+ can exhibit the most pronounced fluorescence response of the polymer Hg2+ can exhibit the most pronounced fluorescence response of the polymer due to triazole moiety in the polymer main chain as the metal binding ligand. The results indicate this kind of conjugated polymer with triazole moiety synthesized by click reaction can be used as a selective fluorescence sensor for Hg2+ detection.

Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from Escherichia coli

In studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes. The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474 has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes, whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding of the catalytic mechanism of the enzyme.

Metal complexes of modified cyclen as catalysts for hydrolytic restriction of plasmid DNA

Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV–visible spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322 DNA caused hypochromic effects and shifted the acridine absorption at 360 nm to a longer wavelength. Under biologically relevant conditions (37 °C and pH 7.4), specific transition metal complexes of these compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II). Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand (L 1 > L 2 > L 3) and the nature of metal ion cofactor (Co 3+ > Zn 2+ > Cr 2+ > Ni 2+ > Cu 2+ > Fe 3+).

A Dynamic Model of HIV Integrase Inhibition and Drug Resistance

Human immunodeficiency virus type 1 (HIV-1) integrase is one of three virally encoded enzymes essential for replication and, therefore, a rational choice as a drug target for the treatment of HIV-1-infected individuals. In 2007, raltegravir became the first integrase inhibitor approved for use in the treatment of HIV-infected patients, more than a decade since the approval of the first protease inhibitor (saquinavir, Hoffman La-Roche, 1995) and two decades since the approval of the first reverse transcriptase inhibitor (retrovir, GlaxoSmithKline, 1987). The slow progress toward a clinically effective HIV-1 integrase inhibitor can at least in part be attributed to a poor structural understanding of this key viral protein.Here we describe the development of a restrained molecular dynamics protocol that produces a more accurate model of the active site of this drug target. This model provides an advance on previously described models as it ensures that the catalytic DDE motif makes correct, monodentate interactions with the two active-site magnesium ions. Dynamic restraints applied to this coordination state create models with the correct solvation sphere for the metal ion complex and highlight the coordination sites available for metal-binding ligands. Application of appropriate dynamic flexibility to the core domain allowed the inclusion of multiple conformational states in subsequent docking studies.These models have allowed us to (1) explore the effects of key drug resistance mutations on the dynamic flexibility and conformational preferences of HIV integrase and to (2) study raltegravir binding in the context of these dynamic models of both wild type and the G140S/Q148H drug-resistant enzyme.

Luminescence of terbium and europium ions in metal-polymer complexes based on poly(amido acids) with main-chain arylcarboxyamide groups

Poly(amido acids) with main-chain metal-binding ligands methylene-bis(anthranilic acid) and their linking fragments of a widely varied structure have been synthesized. The chemical structure of poly(amido acids) that affect the luminescence of Tb3+ and Eu3+ ions and interact with them is studied, and the mechanism of this effect is advanced.

The Missing Link in Plant Histidine Biosynthesis: Arabidopsismyoinositol monophosphatase-like2Encodes a Functional Histidinol-Phosphate Phosphatase

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

A Novel Method of 18F Radiolabeling for PET

Small biomolecules are typically radiolabeled with (18)F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein (18)F is first attached to aluminum as Al(18)F, which is then bound to a chelate attached to a peptide, forming a stable Al(18)F-chelate-peptide complex in an efficient 1-pot process. For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl(3).6H(2)O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of (18)F to form the Al(18)F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH(2)) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100 degrees C for 15 min. In vitro and in vivo stability and targeting ability of the Al(18)F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). The radiolabeled peptide was produced in 5%-20% yield with an estimated specific activity of 18,500-48,100 GBq (500-1,300 Ci)/mmol. The Al(18)F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al(18)F had higher tissue uptake, particularly in the bones, than the chelated Al(18)F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al(18)F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. The ability to bind highly stable Al(18)F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

Metal-Binding Dependent Disruption of Membranes by Designed Helices

The de novo design of molecular switching peptides is of increasing interest because it tests and extends our fundamental understanding of this process while laying the groundwork for the creation of new chemical and biological sensors. Here, an alpha-helical amphiphilic cell-lytic peptide, mastoparan X, was engineered to bind divalent cations. Binding of Zn(II) or Ni(II) to the designed peptide Mst-HH stabilizes the lytic amphiphilic structure and increases the activity of the peptide. Although both Zn(II) and Ni(II) activate Mst-HH for membrane lysis, they appear to do so via different mechanisms. Additionally, a series of metal binding-site mutants were synthesized to assess the relationship of charge and helical propensity to the toxicity and switchability. Additionally, by changing the characteristics of the metal-binding ligands, we can vary the selectivity of the site.

116. A novel moderate-affinity metal binding ligand confers neuroprotection against 6-hydroxydopamine mediated chronic cell death and improves rotational behavior in a mouse model of parkinson’s disease

116. A novel moderate-affinity metal binding ligand confers neuroprotection against 6-hydroxydopamine mediated chronic cell death and improves rotational behavior in a mouse model of parkinson’s disease

Assessment of ligand binding residue predictions in CASP8

Here we detail the assessment process for the binding site prediction category of the eighth Critical Assessment of Protein Structure Prediction experiment (CASP8). Predictions were only evaluated for those targets that bound biologically relevant ligands and were assessed using the Matthews Correlation Coefficient. The results of the analysis clearly demonstrate that three predictors from two groups (Lee and Sternberg) stand out from the rest. A further two groups perform well over subsets of metal binding or nonmetal ligand binding targets. The best methods were able to make consistently reliable predictions based on model structures, though it was noticeable that the two targets that were not well predicted were also the hardest targets. The number of predictors that submitted new methods in this category was highly encouraging and suggests that current technology is at the level that experimental biochemists and structural biologists could benefit from what is clearly a growing field.

Chemistry of Marine Ligands and Siderophores

Marine microorganisms are presented with unique challenges to obtain essential metal ions required to survive and thrive in the ocean. The production of organic ligands to complex transition metal ions is one strategy to both facilitate uptake of specific metals, such as iron, and to mitigate the potential toxic effects of other metal ions, such as copper. A number of important trace metal ions are complexed by organic ligands in seawater, including iron, cobalt, nickel, copper, zinc, and cadmium, thus defining the speciation of these metal ions in the ocean. In the case of iron, siderophores have been identified and structurally characterized. Siderophores are low molecular weight iron-binding ligands produced by marine bacteria. Although progress has been made toward the identity of in situ iron-binding ligands, few compounds have been identified that coordinate the other trace metals. Deciphering the chemical structures and production stimuli of naturally produced organic ligands and the organisms they come from is fundamental to understanding metal speciation and bioavailability. The current evidence for marine ligands, with an emphasis on siderophores, and discussion of the importance and implications of metal-binding ligands in controlling metal speciation and cycling within the world's oceans are presented.

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.

Amelioration of municipal sludge by Pistia stratiotes L.: Role of antioxidant enzymes in detoxification of metals

Pistia stratiotes when exposed to mixture of municipal sludge and effluent accumulated substantial amount of metals in the fronds and roots. With the increase in the metal accumulation by the plants, a reduction in the concentration of metals was found in leachates. The treated plants showed reduced level in chlorophylls but enhanced level of carotenoids and protein. The plant showed a concomitant increase in the activities of antioxidant enzymes; superoxide dismutase, guiacol peroxidase and also an enhanced level of lipid peroxidation. The activities were more in the root tissues as compared to frond. Initially a reduced level of cysteine content in roots of sludge treated plant as compared to control was found, but with time duration it increased progressively. The level of non-protein thiols also increased gradually at all the durations in both fronds and roots. Thus, beside the reduction of metals from municipal sludge, the ability of P. stratiotes to combat metal generated damages by induced synthesis of antioxidant enzymes and other metal binding ligands shows its suitability for the phytoremediation of the waste.

His-tags as Zn(II) binding motifs in a protein-based fluorescent sensor

Fluorescent indicators that allow real-time imaging of Zn(II) in living cells are invaluable tools for understanding Zn(II) homeostasis. Genetically encoded sensors based on fluorescence resonance energy transfer between fluorescent protein domains have important advantages over synthetic probes. We discovered that hexahistidine tags have a strong tendency to dimerize upon binding of Zn(II) in solution and we used this principle to develop a new protein-based sensor for Zn(II). Enhanced cyan and yellow fluorescent proteins were connected by long flexible peptide linkers and His-tags were incorporated at both termini of this fusion protein. The resulting sensor CLY9-2His allows the ratiometric fluorescent detection of Zn(II) in the nanomolar range. In addition, CLY9-2His is selective over the physiologically relevant metal ions Fe(II), Mn(II), Ca(II) and Mg(II). Our approach demonstrates the potential of using small peptides as metal-binding ligands in chelating fluorescent protein chimeras.

Design, synthesis, and metal binding of novel Pseudo‐ oligopeptides containing two phosphinic acid groups

Phosphinic compounds have potential as amide-bond mimetics in the development of novel peptidomimetics, enzyme inhibitors, and metal-binding ligands. Novel pseudo-oligopeptides with two phosphinic acid groups embedded in the peptide backbone serving as amide-bond surrogates, Psi[P(O,OH)--CH(2)], were targeted. A series of linear and cyclic pseudo-oligopeptides with two phosphinic acid groups arrayed at different positions in the peptide sequence were designed, including Ac--Phe--{(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly}(2)--NH(2) (P2), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P3), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe--(R,S) --AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P4), cyclo{NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe}(2) (P5), and cyclo[NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe](2) (P6). They were synthesized via conventional Fmoc chemistry on solid support utilizing Fmoc-protected phosphinic acid-containing pseudo-dipeptide fragment, i.e. Fmoc--(R,S)--AlaPsi[P(O,OCH(3))--CH(2)]Gly--OH. The pseudo-peptides containing two phosphinic acid groups exhibited the highest binding affinity and selectivity for Fe(III) among the 10-metal ions screened by ESI-MS analysis--Cu(II), Zn(II), Co(II), Ni(II), Mn(II), Fe(II), Fe(III), Al(III), Ga(III), and Gd(III). P4 and P6 with 11-atom linkages between the two phosphinic acids preferred intramolecular metal binding to form 1:1 ligand/metal complexes. As revealed by competition experiments, P4 showed the highest relative binding affinity among the six compounds tested. Noteworthy, P4 also showed higher relative binding affinity than similar dihydroxamate-containing pseudo-peptides reported previously. The novel structural prototype and facile synthesis along with selective and potent Fe(III) binding strongly suggest that pseudo-peptides containing the two or more phosphinic groups as amide-bond surrogates deserve further exploration in medicinal chemistry.

Strong Colloidal and Dissolved Organic Ligands Binding Copper and Zinc in Rivers

The speciation or physicochemical form of copper and zinc in freshwater plays an important role in reactivity, bioavailability, and toxicity. Strong metal-binding ligands, which determine speciation, were detected by voltammetric methods, both anodic stripping voltammetry (ASV) and competitive ligand equilibration adsorptive stripping voltammetry (CLE-AdSV); the latter technique can detect nanomolar levels of extremely strong (log K' > 13) ligands. Through careful field site selection and the investigation of ultrafiltration permeate samples, natural organic ligands were measured with limited interferences of colloidal inorganic iron- and aluminum-based trace metal-binding phases. Furthermore, ultrafiltration allowed measurement of colloidal and dissolved ligands independently, and differences of ligand abundance and strength in different size classes are reported. For copper, ultrafilterable (<3 kDa) organic ligand site concentrations (expressed normalized to dissolved organic carbon) were on average 33% of the colloidal level, but ultrafilterable ligand log K' values were 0.5 log units stronger than those of the 0.4 microm filterable concentration. The ultrafilterable copper-binding ligand concentration showed a smaller variation across the rivers (25% rsd) than zinc-binding ligands (90% rsd). For all field sites and size fractions, strong ligand sites greatly exceeded metal concentrations; subsequently, equilibrium speciation modeling predict picomolar levels of free metal. Modeling also indicated that the very strong ligands (detected by CLE-AdSV) predominate, so modeling based solely on ASV data in freshwater may be inadequate. Competition experiments indicated that the very strong ligand sites are metal specific for copper and zinc.

Analysis of glutathione endpoints for measuring copper stress in Chlamydomonas reinhardth

Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d(-1). The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints.

Metal ligand substitution and evidence for quinone formation in taurine/α-ketoglutarate dioxygenase

The three metal-binding ligands of the archetype Fe II/α-ketoglutarate (αKG)-dependent hydroxylase, taurine/αKG dioxygenase (TauD), were systematically mutated to examine the effects of various ligand substitutions on enzyme activity and metallocenter properties. His99, coplanar with αKG and Fe II, is unalterable in terms of maintaining an active enzyme. Asp101 can be substituted only by a longer carboxylate, with the D101E variant exhibiting 22% the k cat and threefold the K m of wild-type enzyme. His255, located opposite the O 2-binding site, is less critical for activity and can be substituted by Gln or even the negatively charged Glu (81% and 33% active, respectively). Transient kinetic studies of the three highly active mutant proteins reveal putative Fe IV-oxo intermediates as reported in wild-type enzyme, but with distinct kinetics. Supplementation of the buffer with formate enhances activity of the D101A variant, consistent with partial chemical rescue of the missing metal ligand. Upon binding Fe II, anaerobic samples of wild-type TauD and the three highly active variants generate a weak green chromophore resembling a catecholate–Fe III species. Evidence is presented that the quinone oxidation state of dihydroxyphenylalanine, formed by aberrant self-hydroxylation of a protein side chain of TauD during aerobic bacterial growth, reacts with Fe II to form this species. The spectra associated with Fe II–TauD and Co II–TauD in the presence of αKG and taurine were examined for all variants to gain additional insights into perturbations affecting the metallocenter. These studies present the first systematic mutational analysis of metallocenter ligands in an Fe II/αKG-dependent hydroxylase.