Non-heme Iron Oxidase Research Articles

Directed evolution of non-heme iron enzymes to access a non-natural radical-relay C(sp3)−H azidation

Recently in Science, Huang and co-workers applied directed evolution for the improvement of a non-heme iron oxidase to catalyze the enantioselective radical-relay azidation of N-fluoroamide substrates. They demonstrated the synthetic applicability by using the improved variant in a one-pot, two-step chemoenzymatic synthesis of a highly functionalized triazole product.

Isopenicillin N Synthase: Crystallographic Studies.

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase (NHIO) that catalyses the cyclisation of tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN). Over the last 25 years, crystallography has shed considerable light on the mechanism of IPNS catalysis. The first crystal structure, for apo-IPNS with Mn bound in place of Fe at the active site, reported in 1995, was also the first structure for a member of the wider NHIO family. This was followed by the anaerobic enzyme-substrate complex IPNS-Fe-ACV (1997), this complex plus nitric oxide as a surrogate for co-substrate dioxygen (1997), and an enzyme product complex (1999). Since then, crystallography has been used to probe many aspects of the IPNS reaction mechanism, by crystallising the protein with a diversity of substrate analogues and triggering the oxidative reaction by using elevated oxygen pressures to force the gaseous co-substrate throughout protein crystals and maximise synchronicity of turnover in crystallo. In this way, X-ray structures have been elucidated for a range of complexes closely related to and/or directly derived from key intermediates in the catalytic cycle, thereby answering numerous mechanistic questions that had arisen from solution-phase experiments, and posing many new ones. The results of these crystallographic studies have, in turn, informed computational experiments that have brought further insight. These combined crystallographic and computational investigations augment and extend the results of earlier spectroscopic analyses and solution phase studies of IPNS turnover, to enrich our understanding of this important protein and the wider NHIO enzyme family.

Mechanism of fatty acid decarboxylation catalyzed by a non-heme iron oxidase (UndA): a QM/MM study.

UndA is a non-heme iron enzyme that was recognized to catalyze the decarboxylation of medium chain (C10-C14) fatty acids to produce trace amounts of 1-alkenes. Owing to the electron imbalance during the oxidative decarboxylation of the substrate and the reduction of O2, only single turnover reactions were obtained in UndA in vitro assays. Unlike the general non-heme iron enzymes, the catalytic efficiency of UndA is quite low. According to the previous proposal, both FeIII-OO˙- and FeIV[double bond, length as m-dash]O complexes may abstract the β-H of fatty acids to trigger the oxidative decarboxylation reaction. Herein, on the basis of the crystal structures of UndA in complex with the substrate analogues, we constructed a series of computational models and performed quantum mechanics/molecular mechanics (QM/MM) calculations to explore the UndA-catalyzed decarboxylation using lauric acid as the substrate. Our calculation results reveal that only the FeIII-OO˙- complex can initiate the decarboxylation, and the substrate (lauric acid) should monodentately coordinate to the Fe center to facilitate the β-H abstraction. In addition, the monodentate coordination corresponds to higher relative energy than the bidentate mode, which may explain the low efficiency of UndA. It is also revealed that as long as the β-H is extracted by the FeIII-OO˙-, the decarboxylation of the substrate radical is quite easy, and an electron transfer from the substrate to the iron center is the prerequisite. For the FeIV[double bond, length as m-dash]O complex, since the β-H is far from the OFe atom and the angle of ∠Fe-O-H is 53.1°, the H-abstraction is calculated to be difficult.

Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6β-Hydroxylase.

Hyoscyamine 6β-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.

High-spin iron(II) complexes of halides and pseudo-halides with biphenyl-appended N,N′-bidentate ligand: X-ray structural and spectroscopic studies

Treatment of the novel biphenyl-appended N,N′-bidentate ligand, 6-(2-pyridylmethyl)-6,7-dihydro-5H-dibenzo[c,e]azepine with FeX2 (X=Cl-, Br−) affords two stable four-coordinate, pseudo-tetrahedral [Fe(NN′)X2] (where X=Cl−, 1; Br−, 2) complexes. In contrast, the same reaction in the presence of pseudo-halides, KNCS/Se, gives two six-coordinate distorted-octahedral, [trans-Fe(NN′)2(NCE)2] (where E=S, 3; Se, 4) complexes. All the four complexes were characterized by X-ray crystallography, spectroscopic (FTIR, Uv–Vis, paramagnetic 1H NMR, Mossbauer of 1) methods, ESI-MS and variable-temperature magnetic susceptibility measurements (VSM, 20–300K). The molecules in the crystal lattice are held together by several strong intermolecular hydrogen-bonding, π⋯π stacking (py-py or py-ph) and C–H⋯π (CH2-ph) interactions. The strong absorption spectral features of 3 and 4 in solution and the paramagnetic 1H NMR spectra of 1–4 are interpreted. Variable-temperature magnetic susceptibility measurements of solids 1–4 show magnetic moment values near 4.9 B.M., characteristic of temperature-independent high-spin (S=2) state for Fe(II). Mossbauer spectrum of 1 complements this property. The monomeric nature of 1 and 2 is an attractive feature, which may serve as invaluable precursor to model the active-site structure and function of mononuclear non-heme iron oxidase (MNO) enzymes, involved in O2 activation and oxidation catalysis of aromatic organic substrates.

The molecular steps of citrinin biosynthesis in fungi.

The individual steps of citrinin 1 biosynthesis in Monascus ruber M7 were determined by a combination of targeted gene knockout and heterologous gene expression in Aspergillus oryzae. The pathway involves the synthesis of an unreduced trimethylated pentaketide 10 by a non-reducing polyketide synthase (nrPKS) known as CitS. Reductive release yields the keto-aldehyde 2 as the first enzyme-free intermediate. The nrPKS appears to be assisted by an as-yet cryptic hydrolysis step catalysed by CitA which was previously wrongly annotated as an oxidase. CitB is a non-heme iron oxidase which oxidises the 12-methyl of 2 to an alcohol. Subsequent steps are catalysed by CitC which oxidises the 12-alcohol to an aldehyde and CitD which converts the 12-aldehyde to a carboxylic acid. Final reduction of C-3 by CitE yields citrinin. The pathway rules out alternatives involving intramolecular rearrangements, and fully defines the molecular steps for the first time and corrects previous errors in the literature. The activity of CitB links the pathway to fungal tropolone biosynthesis and the observation of aminated shunt products links the pathway to azaphilone biosynthesis. Production of citrinin by coordinated production of CitS + CitA-CitE in the heterologous host A. oryzae, in which each gene was driven by a constitutive promoter, was achieved in high yield.

Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis.

The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr (“FYY”). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide “FYY” as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis.

Characterization of Water Coordination to Ferrous Nitrosyl Complexes with fac-N2O, cis-N2O2, and N2O3 Donor Ligands.

Electron paramagnetic resonance (EPR) experiments were done on a series of S = (3)/2 ferrous nitrosyl model complexes prepared with chelating ligands that mimic the 2-His-1-carboxylate facial triad iron binding motif of the mononuclear nonheme iron oxidases. These complexes formed a comparative family, {FeNO}(7)(N2Ox)(H2O)3-x with x = 1-3, where the labile coordination sites for the binding of NO and solvent water were fac for x = 1 and cis for x = 2. The continuous-wave EPR spectra of these three complexes were typical of high-spin S = (3)/2 transition-metal ions with resonances near g = 4 and 2. Orientation-selective hyperfine sublevel correlation (HYSCORE) spectra revealed cross peaks arising from the protons of coordinated water in a clean spectral window from g = 3.0 to 2.3. These cross peaks were absent for the {FeNO}(7)(N2O3) complex. HYSCORE spectra were analyzed using a straightforward model for defining the spin Hamiltonian parameters of bound water and showed that, for the {FeNO}(7)(N2O2)(H2O) complex, a single water conformer with an isotropic hyperfine coupling, Aiso = 0.0 ± 0.3 MHz, and a dipolar coupling of T = 4.8 ± 0.2 MHz could account for the data. For the {FeNO}(7)(N2O)(H2O)2 complex, the HYSCORE cross peaks assigned to coordinated water showed more frequency dispersion and were analyzed with discrete orientations and hyperfine couplings for the two water molecules that accounted for the observed orientation-selective contour shapes. The use of three-pulse electron spin echo envelope modulation (ESEEM) data to quantify the number of water ligands coordinated to the {FeNO}(7) centers was explored. For this aspect of the study, HYSCORE spectra were important for defining a spectral window where empirical integration of ESEEM spectra would be the most accurate.

Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase.

Aliphatic medium-chain 1-alkenes (MCAEs, ∼10 carbons) are "drop-in" compatible next-generation fuels and precursors to commodity chemicals. Mass production of MCAEs from renewable resources holds promise for mitigating dependence on fossil hydrocarbons. An MCAE, such as 1-undecene, is naturally produced by Pseudomonas as a semivolatile metabolite through an unknown biosynthetic pathway. We describe here the discovery of a single gene conserved in Pseudomonas responsible for 1-undecene biosynthesis. The encoded enzyme is able to convert medium-chain fatty acids (C10-C14) into their corresponding terminal olefins using an oxygen-activating, nonheme iron-dependent mechanism. Both biochemical and X-ray crystal structural analyses suggest an unusual mechanism of β-hydrogen abstraction during fatty acid substrate activation. Our discovery unveils previously unidentified chemistry in the nonheme Fe(II) enzyme family, provides an opportunity to explore the biology of 1-undecene in Pseudomonas, and paves the way for tailored bioconversion of renewable raw materials to MCAE-based biofuels and chemical commodities.

The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the β-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.

Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe(II) :ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide δ- l-α-aminoadipoyl- l-cysteinyl- d-valine ( lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. δ- l-α-Aminoadipoyl- l-cysteinyl- d-2-amino-3,3-dideuteriobutyrate ( lld-AC d 2Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer δ- l-α-aminoadipoyl- l-cysteinyl- l-2-amino-3,3-dideuteriobutyrate ( lll-AC d 2Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide δ- l-α-aminoadipoyl- l-cysteinyl- d-2-aminobutyrate ( lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II): lld-AC d 2Ab and IPNS:Fe(II): lll-AC d 2Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.

Design and synthesis of a tetradentate ‘3-amine-1-carboxylate’ ligand to mimic the metal binding environment at the non-heme iron(ii) oxidase active site

Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

The crystal structure of anlll-configured depsipeptide substrate analogue bound to isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(ii) oxidase, which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV) in a remarkable oxidative bicyclisation reaction. The natural substrate for IPNS is the lld-configured tripeptide. lll-ACV is not turned over by the enzyme, but inhibits turnover of the lld-tripeptide. The mechanism by which this inhibition takes place is not fully understood. Recent studies have employed a range of lld-configured depsipeptide substrate analogues in crystallographic studies to probe events preceding beta-lactam closure in the IPNS reaction cycle. Herein, we report the first crystal structure of IPNS in complex with an lll-configured depsipeptide analogue, delta-l-alpha-aminoadipoyl-l-cysteine (1-(R)-carboxy-2-thiomethyl)ethyl ester (lll-ACOmC). This report describes the crystal structure of the IPNS:Fe(ii):lll-ACOmC complex to 2.0 A resolution, and discusses attempts to oxygenate this complex at high pressure in order to probe the mechanism by which lll-configured substrates inhibit IPNS catalysis.

Cis-Dihydroxylation of Alkenes by a Non-Heme Iron Enzyme Mimic

Using the non-heme iron oxidase active site as a template, a peptidomimetic ligand has been designed, synthesized, and used to effect the dihydroxylation ofalkene substrates. Fenton-type radical pathways are also observed.

Isopenicillin N Synthase Mediates Thiolate Oxidation to Sulfenate in a Depsipeptide Substrate Analogue: Implications for Oxygen Binding and a Link to Nitrile Hydratase?

Isopenicillin N synthase (IPNS) is a nonheme iron oxidase that catalyzes the central step in the biosynthesis of beta-lactam antibiotics: oxidative cyclization of the linear tripeptide delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). The ACV analogue delta-L-alpha-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl ester (ACOmC) has been synthesized as a mechanistic probe of IPNS catalysis and crystallized with the enzyme. The crystal structure of the anaerobic IPNS/Fe(II)/ACOmC complex was determined to 1.80 A resolution, revealing a highly congested active site region. By exposing these anaerobically grown crystals to high-pressure oxygen gas, an unexpected sulfenate product has been observed, complexed to iron within the IPNS active site. A mechanism is proposed for formation of the sulfenate-iron complex, and it appears that ACOmC follows a different reaction pathway at the earliest stages of its reaction with IPNS. Thus it seems that oxygen (the cosubstrate) binds in a different site to that observed in previous studies with IPNS, displacing a water ligand from iron in the process. The iron-mediated conversion of metal-bound thiolate to sulfenate has not previously been observed in crystallographic studies with IPNS. This mode of reactivity is of particular interest when considered in the context of another family of nonheme iron enzymes, the nitrile hydratases, in which post-translational oxidation of two cysteine thiolates to sulfenic and sulfinic acids is essential for enzyme activity.

Interactions of Isopenicillin N Synthase with Cyclopropyl-Containing Substrate Analogues Reveal New Mechanistic Insight,

Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.

Substrate-induced conformational changes in Escherichia coli taurine/alpha-ketoglutarate dioxygenase and insight into the oligomeric structure.

The enzymes in the alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily represent the largest class of non-heme iron oxidases and have important medical, ecological, and biotechnological roles. One such enzyme, taurine/alpha-ketoglutarate dioxygenase (TauD), catalyzes the conversion of 2-aminoethanesulfonate (taurine) to sulfite and aminoacetaldehyde while decomposing alphaKG to succinate and CO(2). This alphaKG dependent dioxygenase is expressed in Escherichia coli under sulfur starvation conditions and allows the cell to utilize taurine, and other similar sulfonates in the environment, as an alternative sulfur source. In this work, we report the structures of the apo and holo forms of TauD to 1.9 A resolution (R(cryst) = 21.2%, R(free) = 24.9%) and 2.5 A resolution (R(cryst) = 22.5%, R(free) = 27.8%), respectively. The models reported herein provide significant new insight into the substrate orientations at the active site and the conformational changes that are induced upon taurine binding. Furthermore, analysis of our crystallographic data coupled with reanalysis of the crystallographic model (resolution = 3.0 A, R(cryst) = 28.1, R(free) = 32.0) presented by Elkins et al. (Biochemistry (2002) 41, 5185-5192) reveals an alternative oligomeric arrangement for the enzyme that is consistent with the conserved primary and secondary structure elements of other alphaKG dependent dioxygenases.

Monoanionic N,N,O-Scorpionate Ligands and their Iron(II) and Zinc(II) Complexes: Models for Mononuclear Active Sites of Non-Heme Iron Oxidases and Zinc Enzymes

The chemical behavior of two bis(3,5-dialkylpyrazol-1-yl)acetic acids and their coordination properties towards ZnCl2 and FeCl2 was studied. Reaction of bis(3,5-dimethylpyrazol-1-yl)acetic acid (bdmpza) (1) with ZnCl2 and FeCl2 gave the 2:1 complexes [(bdmpza)2Zn] (4) and [(bdmpza)2Fe] (5). Crystal structures of both complexes were obtained. In contrast, the sterically more hindered ligand bis(3,5-di-tert-butylpyrazol-1-yl)acetic acid (bdtbpza) (3) coordinates only once to zinc(II) resulting in the complex [(bdtbpza)ZnCl] (6). This provides a structural model complex for the active sites of zinc enzymes such as thermolysin or carboxypeptidase. The good agreement with these active sites was demonstrated by a crystal structure of 6. To the best of our knowledge, this is the first report of zinc complexes with a monoanionic N,N,O-tridentate ligand using a carboxylate O-donor. Substitution of benzylthiolate for the chloride ligand 6 yielded [(bdtbpza)Zn(SCH2Ph)] (8) indicating that the bulky tert-butyl groups do not prevent access to the zinc atom. Reaction of bdtbpza (3) with FeCl2 led to [(bdtbpza)FeCl] (7), which might serve as a structural model complex for the active sites of mononuclear non-heme iron oxidases and oxygenases such as IPNS, DAOCS or CAS.

MonoanionicN,N,O-Scorpionate Ligands and their Iron(II) and Zinc(II) Complexes: Models for Mononuclear Active Sites of Non-Heme Iron Oxidases and Zinc Enzymes

The chemical behavior of two bis(3,5-dialkylpyrazol-1-yl)acetic acids and their coordination properties towards ZnCl2 and FeCl2 was studied. Reaction of bis(3,5-dimethylpyrazol-1-yl)acetic acid (bdmpza) (1) with ZnCl2 and FeCl2 gave the 2:1 complexes [(bdmpza)2Zn] (4) and [(bdmpza)2Fe] (5). Crystal structures of both complexes were obtained. In contrast, the sterically more hindered ligand bis(3,5-di-tert-butylpyrazol-1-yl)acetic acid (bdtbpza) (3) coordinates only once to zinc(II) resulting in the complex [(bdtbpza)ZnCl] (6). This provides a structural model complex for the active sites of zinc enzymes such as thermolysin or carboxypeptidase. The good agreement with these active sites was demonstrated by a crystal structure of 6. To the best of our knowledge, this is the first report of zinc complexes with a monoanionic N,N,O-tridentate ligand using a carboxylate O-donor. Substitution of benzylthiolate for the chloride ligand 6 yielded [(bdtbpza)Zn(SCH2Ph)] (8) indicating that the bulky tert-butyl groups do not prevent access to the zinc atom. Reaction of bdtbpza (3) with FeCl2 led to [(bdtbpza)FeCl] (7), which might serve as a structural model complex for the active sites of mononuclear non-heme iron oxidases and oxygenases such as IPNS, DAOCS or CAS.