Aminoquinoline-Based Tridentate (NNN)-Copper Catalyst for C-N Bond-Forming Reactions from Aniline and Diazo Compounds.

A new tridentate Cu2+ complex based on (E)-1-(pyridin-2-yl)-N-(quinolin-8-yl)methanimine (PQM) was generated and characterized to support the activation of diazo compounds for the formation of new C-N bonds. This neutral Schiff base ligand was structurally characterized to coordinate with copper(II) in an equatorial fashion, yielding a distorted octahedral complex. Upon characterization, this copper(II) complex was used to catalyze an efficient and cost-effective protocol for C-N bond formation between N-nucleophiles and copper carbene complexes arising from the activation of diazo carbonyl compounds. A substrate scope of approximately 15 different amine-based substrates was screened, yielding 2° or 3° amine products with acceptable to good yields under mild reaction conditions. Reactivity towards phenol and thiophenol were also screened, showing relatively weak C-O or C-S bond formation under optimized conditions.

Sodium Trifluoroacetate mediated Copper-Catalyzed aza-Michael addition of α,ß-unsaturated olefins with aromatic amines.

We present a sodium trifluoroacetate (CF3CO2Na) mediated copper-catalyzed aza-Michael addition of aromatic amines with activated olefins under mild, aqueous reaction conditions. This simplistic protocol employs a copper catalyst (10 mol%) and water as solvent. This transformation occurs precisely with aromatic substituted amines containing both electron-donating (EDG) and electron-withdrawing (EWG) groups. A broad range of substrates were tested under the optimized conditions, which are producing good to moderate yields.

Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O2 to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O2-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O2 activation. Exploring the energetics of each step before O2 activation can provide mechanistic insight into the initial steps that support the highly specific O2 activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheH·Fe increased the stability of this enzyme (ΔTm of 8.5 (±0.7) °C with an associated δΔH of 43.0 (±2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C-H activation in this enzyme.

Metal Ion Binding Induces Local Protein Unfolding and Destabilizes Human Carbonic Anhydrase II.

Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA binds one zinc ion or two copper ions. We studied these binding processes at five temperatures (15-35 °C) using isothermal titration calorimetry, yielding thermodynamic parameters corrected for pH and buffer effects. We then sought to identify binding-induced structural changes. Our data suggest that binding at the active site organizes 6-8 residues; however, copper binding near the N-terminus results in a net unfolding of 6-7 residues. This surprising destabilization was confirmed using circular dichroism and protein stability measurements. Metal binding induced unfolding may represent an important regulatory mechanism, but it may be easily missed by NMR and X-ray crystallography. Thus, in addition to highlighting a highly novel binding-induced unfolding event, we demonstrate the value of calorimetry for studying the structural implications of metal binding.

Cu-Catalyzed Chan-Evans-Lam Coupling Reactions of 2-Nitroimidazole with Aryl Boronic Acids: An Effort toward New Bioactive Agents against S.†pneumoniae.

The coupling of phenylboronic acids with poorly-activated imidazoles is studied as a model system to explore the use of copper-catalyzed Chan-Evans-Lam (CEL) coupling for targeted C-N bond forming reactions. Optimized CEL reaction conditions are reported for four phenanthroline-based ligand systems, where the ligand 4,5-diazafluoren-9-one (dafo, L2) with 1 molar equivalent of potassium carbonate yielded the highest reactivity. The substrate 2-nitroimidazole (also known as azomycin) has documented antimicrobial activity against a range of microbes. Here N-arylation of 2-nitroimidazole with a range of aryl boronic acids has been successfully developed by copper(II)-catalyzed CEL reactions. Azomycin and a range of newly arylated azomycin derivatives were screened against S. pneumoniae, where 1-(4-(benzyloxy)phenyl)-2-nitro-1H-imidazole (3d) was demonstrated to have a minimal inhibition concentration value of 3.3 µg/mL.

Tuning the copper(II)/copper(I) redox potential for more robust copper-catalyzed C-N bond forming reactions.

Complexes of copper and 1,10-phenanthroline have been utilized for organic transformations over the last 50 years. In many cases these systems are impacted by reaction conditions and perform best under an inert atmosphere. Here we explore the role the 1,10-phenanthroline ligand plays on the electronic structure and redox properties of copper coordination complexes, and what benefit related ligands may provide to enhance copper-based coupling reactions. Copper(II) triflate complexes bearing 1,10-phenanthroline (phen), ([Cu(phen)2(OTf)]OTf, 1) and oxidized derivatives of phen including [Cu(edhp)2](OTf)2 (2), [Cu(pdo)2](OTf)2 (3), [Cu(dafo)2](OTf)2 (4) were prepared and characterized. X-ray crystallographic data show these related ligands subtly impacted the coordination geometry of the copper(II) ion. Complexes 1-3 had only incremental changes to the redox properties of the copper ions, complex 4 showed a drastically different redox potential affording a remarkably air stable copper(I) complex. These complexes 1-4 were then used to catalyze the C-N bond forming cross coupling between imidazole and various boronic acid substrates, where the increased stability of the copper(I) species in complex 4 appears to better support these CEL cross couplings.

Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE), like many other 2-oxoglutarate (2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative decarboxylation of 2OG to succinate and CO2 to generate a highly reactive iron species that hydroxylates a specific alkane C-H bond, in this case targeting l-arginine (Arg) for hydroxylation. However, the prominently observed reactivity of EFE is the transformation of 2OG into ethylene and three molecules of CO2. Crystallographic and biochemical studies have led to several proposed mechanisms for this 2-fold reactivity, but the detailed reaction steps are still obscure. Here, the thermodynamics associated with iron(II), 2OG, and Arg binding to EFE are studied using calorimetry (isothermal titration calorimetry and differential scanning calorimetry) to gain insight into how these binding equilibria organize the active site of EFE, which may have an impact on the O2 activation pathways observed in this system. Calorimetric data show that the addition of iron(II), Arg, and 2OG increases the stability over that of the apoenzyme, and there is distinctive cooperativity between substrate and cofactor binding. The energetics of binding of 2OG to Fe·EFE are consistent with a unique monodentate binding mode, which is different than the prototypical 2OG coordination mode in other 2OG-dependent oxygenases. This difference in the pre-O2 activation equilibria may be important for supporting the alternative ethylene-forming chemistry of EFE.

The Irving-Williams series and the 2-His-1-carboxylate facial triad: a thermodynamic study of Mn2+, Fe2+, and Co2+ binding to taurine/α-ketoglutarate dioxygenase (TauD).

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD) is an E. coli nonheme Fe2+- and αKG-dependent metalloenzyme that catalyzes the hydroxylation of taurine, leading to the production of sulfite. The metal-dependent active site in TauD is formed by two histidine and one aspartate that coordinating to one face of an octahedral coordination geometry, known as the 2-His-1-carboxylate facial triad. This motif is found in many nonheme Fe2+ proteins, but there is limited information on the thermodynamic parameters that govern metal-ion binding to this site. Here, we report data from calorimetry and related biophysical techniques to generate complete thermodynamic profiles of Mn2+ and Co2+ binding to TauD, and these values are compared to the Fe2+ data reported earlier Henderson et al. (Inorg Chem 54: 2278-2283, 2015). The buffer-independent binding constants (K) were measured to be 1.6 × 106, 2.4 × 107, and 1.7 × 109, for Mn2+, Fe2+, and Co2+, respectively. The corresponding ΔG° values were calculated to be - 8.4, - 10.1, and - 12.5 kcal/mol, respectively. The metal-binding enthalpy changes (ΔH) for these binding events are - 11.1 (± 0.1), - 12.2 (± 0.1), and - 16.0 (± 0.6) kcal/mol, respectively. These data are fully consistent with the Irving-Williams series, which show an increasing affinity for transition metal ions across the periodic table. It appears that the periodic increase in affinity, however, is a result of a complicated summation of enthalpy terms (including favorable metal-ion coordination processes and unfavorable ionization events) and related entropy terms.

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme-substrate interactions.

BACKGROUND: Extradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step. METHODS: This study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments. RESULTS: The equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2×10(4) to 1×10(6), suggesting there are distinctive differences in how the enzyme-substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts. CONCLUSIONS: Condition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme-substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity. GENERAL SIGNIFICANCE: Substrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation.

Calorimetric and spectroscopic investigations of the binding of metallated porphyrins to G-quadruplex DNA.

BACKGROUND: The human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure. METHODS: Isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na(+)] or [K(+)]≈0.15M). RESULTS: The apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a "four-independent-sites model". The two highest-affinity sites exhibit a K in the range of 10(8) to 10(10)M(-1) with the two lower-affinity sites exhibiting a K in the range of 10(4) to 10(5)M(-1). Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a "two-independent-sites model" in which only the end-stacking binding mode is observed with a K in the range of 10(4) to 10(5)M(-1). CONCLUSIONS: In the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an "end stacking" mechanism for the higher affinity binding mode and an "intercalation" mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the "end-stacking" mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands. GENERAL SIGNIFICANCE: The preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex.

Resolving distinct molecular origins for copper effects on PAI-1.

Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator inhibitor-1 (PAI-1), which is responsible for specific and rapid inhibition of fibrinolytic proteases. Active PAI-1 is inherently unstable and readily converts to a latent, inactive form. The binding of vitronectin and other ligands influences stability of active PAI-1. Our laboratory recently observed reciprocal effects on the stability of active PAI-1 in the presence of transition metals, such as copper, depending on the whether vitronectin was also present (Thompson et al. Protein Sci 20:353-365, 2011). To better understand the molecular basis for these copper effects on PAI-1, we have developed a gel-based copper sensitivity assay that can be used to assess the copper concentrations that accelerate the conversion of active PAI-1 to a latent form. The copper sensitivity of wild-type PAI-1 was compared with variants lacking N-terminal histidine residues hypothesized to be involved in copper binding. In these PAI-1 variants, we observed significant differences in copper sensitivity, and these data were corroborated by latency conversion kinetics and thermodynamics of copper binding by isothermal titration calorimetry. These studies identified a copper-binding site involving histidines at positions 2 and 3 that confers a remarkable stabilization of PAI-1 beyond what is observed with vitronectin alone. A second site, independent from the two histidines, binds metal and increases the rate of the latency conversion.

Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes.

BACKGROUND: TauD is a nonheme iron(II) and α-ketoglutarate (αKG) dependent dioxygenase, and a member of a broader family of enzymes that oxidatively decarboxylate αKG to succinate and carbon dioxide thereby activating O2 to perform a range of oxidation reactions. However before O2 activation can occur, these enzymes bind both substrate and cofactor in an effective manner. Here the thermodynamics associated with substrate and cofactor binding to FeTauD are explored. METHODS: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes are explored using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). RESULTS: Taurine binding is endothermic (+26kcal/mol) and entropically driven that includes burial of hydrophobic surfaces to close the lid domain. Binding of αKG is enthalpically favorable and shows cooperativity with taurine binding, where the change in enthalpy associated with αKG binding (δΔHcal) increases from -30.1kcal/mol when binding to FeTauD to -65.2kcal/mol when binding to the FeTauD-taurine complex. CONCLUSIONS: The intermolecular interactions that govern taurine and αKG binding impact the global stability of TauD and its complexes, with clear and dramatic cooperativity between substrate and cofactor. GENERAL SIGNIFICANCE: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes each exhibited increased temperature stability over the free enzyme. Through deconvolution of the energetic profiles for all species studied, a thermodynamic cycle was generated that shows significant cooperativity between substrate and cofactor binding which continues to clarity the events leading up O2 activation.

Calorimetric assessment of Fe(2+) binding to α-ketoglutarate/taurine dioxygenase: ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad.

The thermodynamic properties of Fe(2+) binding to the 2-His-1-carboxylate facial triad in α-ketoglutarate/taurine dioxygenase (TauD) were explored using isothermal titration calorimetry. Direct titrations of Fe(2+) into TauD and chelation experiments involving the titration of ethylenediaminetetraacetic acid into Fe(2+)-TauD were performed under an anaerobic environment to yield a binding equilibrium of 2.4 (±0.1) × 10(7) (Kd = 43 nM) and a ΔG° value of -10.1 (±0.03) kcal/mol. Further analysis of the enthalpy/entropy contributions indicates a highly enthalpic binding event, where ΔH = -11.6 (±0.3) kcal/mol. Investigations into the unfavorable entropy term led to the observation of water molecules becoming organized within the Fe(2+)-TauD structure.

Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II.

Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo form of human carbonic anhydrase II (CA) binds 2 equiv of copper(II) with high affinity. The Cu(2+) ions bind independently forming two noncoupled type II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu(2+) binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four- to five-coordinate N-terminal Cu(2+) binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu(2+) and Ni(2+) and copper(II)-ß-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu(2+) binding data to the high-affinity CuA site, we derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu(2+) binding to the CuB site of CA to be 2 × 10(9) and -17.4 kcal/mol, respectively.

Three water-soluble copper(II) N-heterocyclic carbene complexes: toward copper-catalyzed ketone reduction under sustainable conditions.

A series of tridentate copper(II) N-heterocyclic carbene (NHC) complexes with imidazole, benzimidazole, and 5,6-dimethylbenzimidazole azole rings were synthesized and comprehensively characterized via X-ray crystallography, ESI-MS, cyclic voltammetry, and UV-Vis and EPR spectroscopic studies. These complexes were then utilized for the optimization of ketone reduction under sustainable conditions using 2-acetylpyridine and phenylsilane. The relationships between product formation, temperature, reaction time, and catalyst loading for the hydrogenation reactions are covered in detail. Reduction of eighteen different aliphatic, cyclic, and aromatic ketones were demonstrated, which were compatible to produce the corresponding products in moderate to good yields. These systems were used to develop related DNA-hybrid catalytic systems, but only supported weak enantioselectivity. Further thermodynamic experiments showed Cu-NHC complexes did not demonstrate specific binding to DNA, which is consistent with their limited selectivity.

Building reactive copper centers in human carbonic anhydrase II.

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu(2+) ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu(2+)-binding events have different affinities (K a approximately 5 × 10(12) and 1 × 10(10)), and both are enthalpically driven processes. Reconstituting these Cu(2+) sites under a range of conditions has allowed us to assign the Cu(2+)-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu(2+) sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

Copper complexes for the chemoselective N-arylation of arylamines and sulfanilamides via Chan-Evans-Lam cross-coupling.

Copper(II) complexes with tridentate NNN-ligands were utilized for Chan-Evans-Lam (CEL) cross-coupling reactions to enable the N-arylation of multifarious N-nucleophiles through the activation of aryl boronic acids. A condition-specific methodology was developed to chemoselectively target the amine versus sulfonamide N-arylation of 4-aminobenzenesulfonamide using new catalysts. Two different pyridine-based ligands and corresponding copper(II) complexes were characterized using 1H and 13C-NMR, FTIR, and UV-vis spectroscopy, HRMS, single-crystal X-ray diffraction, and cyclic voltammetry. Solvent and base-controlled cross-coupling reactions were observed, which led to the optimization of selective conditions for targeted C-N bond formation of sulfanilamides. Beyond the chemoselective processes reported here, a breadth of N-nucleophiles including sulfanilamides and arylamines were screened for arylation by this CEL catalyst.

Calorimetric analysis of AdcR and its interactions with zinc(II) and DNA.

Zinc(II) ions play critical roles in all known life as structurally important stabilizing ions in proteins, catalytically active metals in enzymes, and signaling agents impacting physiological changes. To maintain homeostasis, the intracellular concentration of zinc(II) is strictly controlled by a family of metal-regulatory proteins in both prokaryotic and eukaryotic organisms. In S. pneumoniae, there are two proteins that share responsibility for Zn2+ homeostasis, one of them is the Adhesin Competence Repressor (AdcR) and it binds to a specific double-stranded DNA binding domain (dsDNA). AdcR has been structurally characterized containing two zinc(II) metal centers per monomeric unit. Here we report data collected from differential scanning calorimetry (DSC) experiments aimed to measure the structural stability of AdcR, the fully complimented Zn2AdcR complex, and the protein/DNA complex Zn2AdcR/dsDNA. Thermograms collected from DSC experiments yielded endothermic unfolding events for AdcR, Zn2AdcR, and Zn2AdcR/dsDNA complex at 55.6, 70.2, and 56.6 °C, respectively. A non-two state unfolding model best fits the data, giving ΔH terms associated with these thermal unfolding events of 5.1, 7.1, and 4.9 kcal/mol. These data allow for the development of a thermodynamic cycle connecting both zinc(II) and DNA binding to AdcR. Furthermore, pairing this newly reported data with known association constants for zinc(II) and DNA binding allowed for the generation of thermodynamic profiles for both zinc(II) binding to AdcR and Zn2AdcR binding to DNA, which show both are decisively entropy-driven processes.

Exploring substrate binding in homoprotocatechuate 2,3-dioxygenase using isothermal titration calorimetry.

Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K = 1.1 ± 0.6 × 10(6)) and enthalpy change (ΔH = -17.0 ± 1.7 kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.

Revisiting zinc coordination in human carbonic anhydrase II.

Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA, CA where Zn(2+) ion has been removed) is relatively easy to generate, and reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (K(a) ≈ 1.2 × 10(12)) for Zn(2+). However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn(2+) with a strong equilibrium constant (K(a) ≈ 2 × 10(9)) that is 3 orders of magnitude lower than the previously established value. Thermodynamic parameters associated with Zn(2+) binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and the reconstituted ZnCA (active CA containing a mononuclear Zn(2+) center) were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirm the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.