Amy R. Marts

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM‑1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data.

Biochemical, mechanistic, and spectroscopic characterization of metallo-β-lactamase VIM-2.

This study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.

'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn2+ ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a ‘flattened’ mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal–ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal–ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms’ tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)–WT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 M–1, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least −17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)–WT1-4 to those from the model peptide Zn(II)–GGG–Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, −17.6 kcal/mol.

Paramagnetic Resonance of Cobalt(II) Trispyrazolylmethanes and Counterion Association

Paramagnetic resonance studies (EPR, ESEEM, ENDOR, and NMR) of a series of cobalt(II) bis-trispyrazolylmethane tetrafluoroborates are presented. The complexes studied include the parent, unsubstituted ligand (Tpm), two pyrazole-substituted derivatives (4Me and 3,5-diMe), and tris(1-pyrazolyl)ethane (Tpe), which includes a methyl group on the apical carbon atom. NMR and ENDOR establish the magnitude of 1H hyperfine couplings, while ESEEM provides information on the coordinated 14N. The data show that the pyrazole 3-position is more electron rich in the Tpm analogues, that the geometry about the apical atom influences the magnetic resonance, and that apical atom geometry appears more fixed in Tpm than in Tp. NMR and ENDOR establish that the BF4- counterion remains associated in fluid solution. In the case of the Tpm3,5Me complex, it appears to associate in solution, in the same position it occupies in the X-ray structure.

Substituent Effects on the Coordination Chemistry of Metal-Binding Pharmacophores

A combination of XAS, UV-vis, NMR, and EPR was used to examine the binding of a series of α-hydroxythiones to CoCA. All three appear to bind preferentially in their neutral, protonated forms. Two of the three clearly bind in a monodentate fashion, through the thione sulfur alone. Thiomaltol (TM) appears to show some orientational preference, on the basis of the NMR, while it appears that thiopyromeconic acid (TPMA) retains rotational freedom. In contrast, allothiomaltol (ATM), after initially binding in its neutral form, presumably through the thione sulfur, forms a final complex that is five-coordinate via bidentate coordination of ATM. On the basis of optical titrations, we speculate that this may be due to the lower initial pKa of ATM (8.3) relative to those of TM (9.0) and TPMA (9.5). Binding through the thione is shown to reduce the hydroxyl pKa by ∼0.7 pH unit on metal binding, bringing only ATMs pKa close to the pH of the experiment, facilitating deprotonation and subsequent coordination of the hydroxyl. The data predict the presence of a solvent-exchangeable proton on TM and TPMA, and Q-band 2-pulse ESEEM experiments on CoCA + TM suggest that the proton is present. ESE-detected EPR also showed a surprising frequency dependence, giving only a subset of the expected resonances at X-band.

Geometry of Zn(II) complexes with dissolved organic matter: X-ray studies at variable pH

The structure of Zn(II) complexes with dissolved organic matter (DOM) is an important consideration in developing molecular-level models of Zn(II) speciation, but recent reports favoring the tetrahedral geometry differ from earlier findings that geometry was largely octahedral. In general, the presence of thiolate ligands favors the tetrahedral geometry, while O and N ligands favor the octahedral geometry. This work presents extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectroscopic results, indicating an octahedral geometry over the pH range of 5–9 for a freshwater DOM isolate. Changes in XANES derivatives as a function of pH can be explained in terms of ligand protonation and/or changing ligand groups. Tetrahedral Zn(II)–DOM geometry may be restricted to binding environments containing thiol groups.