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• W2022172512 abstract "d- and l-captopril are competitive inhibitors of metallo-β-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of γ-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-β-lactamase subclasses B1 and B2, we find two different inhibitor binding modes. d- and l-captopril are competitive inhibitors of metallo-β-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of γ-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-β-lactamase subclasses B1 and B2, we find two different inhibitor binding modes. Metallo-β-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of β-lactam antibiotics, including carbapenems. This relatively new form of resistance is spreading and thereby escaping the effective inhibitors developed to fight the better known serine-β-lactamases. For all metallo-β-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-β-lactamase (BcII), 1The abbreviations used are: BcII, metallo-β-lactamase from B. cereus; CphA, metallo-β-lactamase from A. hydrophila; PAC, perturbed angular correlation of γ-rays; EXAFS, extended x-ray absorption fine structure; H-site, zinc binding site composed of three histidine residues in subclass B1; DCH site, zinc binding site composed of asparagine, cysteine, and histidine; NQI, nuclear quadrupole interaction; XAS, x-ray absorption spectroscopy. one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1Carfi A. Pares S. Duee E. Galleni M. Duez C. Frere J.M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (403) Google Scholar). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2Massidda O. Rossolini G.M. Satta G. J. Bacteriol. 1991; 173: 4611-4617Crossref PubMed Google Scholar). Various thiol-carboxylate compounds were identified as potent inhibitors (3Payne D.J. Du W. Bateson J.H. Expert. Opin. Investig. Drugs. 2000; 9: 247-261Crossref PubMed Scopus (51) Google Scholar). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4Mollard C. Moali C. Papamicael C. Damblon C. Vessilier S. Amicosante G. Schofield C.J. Galleni M. Frere J.M. Roberts G.C. J. Biol. Chem. 2001; 276: 45015-45023Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), whereas the binding of 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutrylglycine)] to the enzyme from Pseudomonas aeruginosa (IMP-1) was characterized by x-ray crystallography (5Concha N.O. Janson C.A. Rowling P. Pearson S. Cheever C.A. Clarke B.P. Lewis C. Galleni M. Frere J.M. Payne D.J. Bateson J.H. Abdel-Meguid S.S. Biochemistry. 2000; 39: 4288-4298Crossref PubMed Scopus (281) Google Scholar). With both approaches, the inhibited binuclear zinc enzymes were studied. Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6Toney J.H. Hammond G.G. Fitzgerald P.M. Sharma N. Balkovec J.M. Rouen G.P. Olson S.H. Hammond M.L. Greenlee M.L. Gao Y.D. J. Biol. Chem. 2001; 276: 31913-31918Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) or moxalactam and cefoxitin for CphA (7Zervosen A. Valladares M.H. Devreese B. Prosperi-Meys C. Adolph H.W. Mercuri P.S. Vanhove M. Amicosante G. van Beeumen J. Frere J.M. Galleni M. Eur. J. Biochem. 2001; 268: 3840-3850Crossref PubMed Scopus (30) Google Scholar). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products. The structural investigation of d- and gl-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an angiotensin converting enzyme-blocking agent used in the therapy of blood pressure diseases. Although different catalytic mechanisms for mono- and binuclear metallo-β-lactamases have been discussed in the literature, it is still not clearly understood why the enzymes have two conserved metal binding sites (for review, see Ref. 8Wang Z. Fast W. Valentine A.M. Benkovic S.J. Curr. Opin. Chem. Biol. 1999; 3: 614-622Crossref PubMed Scopus (268) Google Scholar). The motivation for the present investigation was the demand for a better knowledge of the nature of metal ion binding in the presence of bound ligands. By a combination of extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of γ-rays (PAC) spectroscopy, we studied the nature of captopril interactions with the cadmium-substituted enzymes (detailed descriptions of the methods can be found in Refs. 9Teo B.K. EXAFS: Basic principles and Data Analysis. Springer, Berlin1986Crossref Google Scholar and 10Paul-Soto R. Zeppezauer M. Adolph H.W. Galleni M. Frere J.M. Carfi A. Dideberg O. Wouters J. Hemmingsen L. Bauer R. Biochemistry. 1999; 38: 16500-16506Crossref PubMed Scopus (33) Google Scholar, respectively). Both methods delivered consistent results that are additionally supported by UV-visible spectroscopic results of Co(II)-substituted enzymes. The present investigation contributes new insights with respect to the physiological importance of mono- and binuclear metallo-β-lactamases. The metallo-β-lactamases CphA from Aeromonas hydrophila AE036 and BcII from B. cereus 569/H/9 were purified as described (11Hernandez Valladares M. Galleni M. Frère J.M. Felici A. Perilli M. Franceschini N. Rossolini G.M. Oratore A. Amicosante G. Microb. Drug Resist. 1996; 2: 253-256Crossref PubMed Scopus (21) Google Scholar, 12Paul-Soto R. Bauer R. Frere J.M. Galleni M. Meyer-Klaucke W. Nolting H. Rossolini G.M. de Seny D. Hernandez-Valladares M. Zeppezauer M. Adolph H.W. J. Biol. Chem. 1999; 274: 13242-13249Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The protein concentrations were determined by measuring the absorbance at 280 nm using extinction coefficients of 30,500 m–1 cm–1 for BcII 569/H/9 and 38,000 m–1 cm–1 for CphA. Metal ion concentrations in samples and in the final dialysis buffers were determined by atomic absorption spectroscopy in the flame mode as described (13Hernandez V.M. Felici A. Weber G. Adolph H.W. Zeppezauer M. Rossolini G.M. Amicosante G. Frere J.M. Galleni M. Biochemistry. 1997; 36: 11534-11541Crossref PubMed Scopus (176) Google Scholar). To produce “metal-free” buffers, buffer solutions in bidistilled water were treated by extensive stirring with Chelex 100 (Sigma). Apo-enzymes were prepared by dialysis of the corresponding enzymes against two changes of 15 mm HEPES, pH 7.0, containing 0.2 m NaCl and 20 mm EDTA over 12 h under stirring. EDTA was removed from the resulting apoenzyme solution by three dialysis steps against the same buffer containing 1 m NaCl and Chelex-100 and finally two dialysis steps against 15 mm HEPES, pH 7.0, containing 0.2 m NaCl and Chelex-100. In all preparations, the residual zinc content did not exceed 5% as determined by atomic absorption spectroscopy. To synthesize d-captopril (Scheme 1), we prepared compound 1 according to the procedure described in Ref. 14Suh J.T. Skiles J.W. Williams B.E. Youssefyeh R.D. Jones H. Loev B. Neiss E.S. Schwab A. Mann W.S. Khandwala A. Wolf P.S. Weinryb I. J. Med. Chem. 1985; 28: 57-66Crossref PubMed Scopus (34) Google Scholar. Compound 2 was prepared following a method reported by Skiles et al. (15Skiles J.W. Suh J.T. Williams B.E. Menard P.R. Barton J.N. Loev B. Jones H. Neiss E.S. Schwab A. J. Med. Chem. 1986; 29: 784-796Crossref PubMed Scopus (48) Google Scholar), and the classical hydrolysis reaction to obtain the d-captopril was carried out with NaOH 1 n under an atmosphere of argon (16Smith E.M. Swiss G.F. Neustadt B.R. Gold E.H. Sommer J.A. Brown A.D. Chiu P.J.S. Moran R. Sybertz E.J. Baum T. J. Med. Chem. 1988; 31: 875-885Crossref PubMed Scopus (70) Google Scholar). As there are two asymmetric centers in the molecule, here d- and l-designations refer to absolute stereochemistry at the prolinyl stereocenter (see Fig. 3).Fig. 3Structural models of the BcII and CphA active sites based on the EXAFS results.A, active site models for both Cd(II)1-BcII and -CphA. B, active site models for both the d-captopril-inhibited species of Cd(II)1-BcII and Cd(II)1-CphA. C, active site model of Zn(II)1-BcII (17de Seny D. Heinz U. Wommer S. Kiefer M. Meyer-Klaucke W. Galleni M. Frere J.M. Bauer R. Adolph H.W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and the two diastereomers d- and l-captopril to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All kinetic measurements were performed at 25 °C with imipenem (kind gift of Merck) as a substrate following the hydrolysis at 300 nm (Δ ϵ (300 nm) = –9000 m–1 cm–1) in 15 mm HEPES, pH 7.0. The photometric measurements were performed with either a spectrophotometer (CphA) or the stopped-flow system DX-17MV (Applied Photophysics, Leatherhead, UK) in those cases where high concentrations of enzymes (0.1–1 μm) during the measurements were required to exactly define the reconstitution state of the metallo-enzymes. Under such conditions, it was possible to study Zn(II)1- and Cd(II)1-BcII with apo-enzyme reconstituted with only 0.1 equivalents of metal without the interference of residual zinc in the metal-depleted buffers. The data evaluation was based on the concentration of metal ions added. Effects of residual zinc in the solutions could be minimized, and it was possible to clearly discriminate Me1 and Me2 species. The steady-state parameters Km and kcat and the inhibition constants for d- and l-captopril were determined from initial rates. Standard non-linear regression analysis was used for data evaluation by directly fitting the Michaelis-Menten equation (uninhibited or competitively inhibited) to the data. Activities of binuclear enzymes were studied with enzyme samples in the presence of excess of the respective metal ions. Inhibition constants for d- and l-captopril were determined by the variation of the inhibitor concentration at substrate concentrations fixed in the range of the respective Km values. UV-visible spectra of Co(II)-substituted BcII were recorded with a Lambda9 spectrophotometer (PerkinElmer Life Sciences) and processed with the UV-Winlab software from PerkinElmer Life Sciences. Co(II)1-BcII was prepared by reconstitution of 130 μm apo-enzyme with 120 μm Co(II). Co(II)2-BcII was prepared by preincubation of 118 μm apo-BcII with 500 μm Co(II). To remove traces of precipitated protein, the samples were centrifuged for 10 min at 30,000 × g immediately before the measurement. The d-captopril complexes were obtained by adding 800 μmd-captopril to the sample cells. The dissociation constants for a first (Kmono) and second (Kbi) cadmium ion bound to BcII in presence of 25 μmd-captopril and 0.1 m NaCl were obtained from competition experiments with the chromophoric chelator Mag-fura-2 (Molecular Probes, Eugene, OR) in 15 mm HEPES, pH 7.0, as described previously (17de Seny D. Heinz U. Wommer S. Kiefer M. Meyer-Klaucke W. Galleni M. Frere J.M. Bauer R. Adolph H.W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Hemmingsen L. Damblon C. Antony J. Jensen M. Adolph H.W. Wommer S. Roberts G.C. Bauer R. J. Am. Chem. Soc. 2001; 123: 10329-10335Crossref PubMed Scopus (48) Google Scholar). X-ray Absorption Spectroscopy (XAS) Sample Preparation—The buffer used during purification has been exchanged for 20 mm bis-Tris, pH 7, by iterative use of Millipore Centricon devices to decrease scattering background. The final protein concentration was ∼2–3 mm. The free metal concentration was below 2 μm. Samples have been frozen and stored at –20 °C. XAS Measurements—For XAS, about 100–120 μl of enzyme solution were transferred to the EXAFS cuvettes covered with Kapton tape (DuPont) as an x-ray transparent window material, capped, mounted on the sample holder, dropped into liquid nitrogen, and transferred to the beamline cryostat. The Cadmium-K edge (26,711.0 eV) XAS was collected at the beamline D2 at Deutsches Elektronen Synchrotron (European Molecular Biology Laboratory outstation, Hamburg, Germany) running at 4.4 GeV and 70–125 mA current in fluorescence mode at 25 K sample temperature. An internal cadmium foil sample was used for calibration. XAS Data Analysis—Standard EXAFS analysis was performed using the EXPROG software package (developed by C. Hermes and H. F. Nolting at the European Molecular Biology Laboratory-Outstation/Hamburg, Germany) to process the raw data and EXCURV98 (developed by N. Binsted, S. W. Cambell, S. J. Gurman, and P. Stepherson at Science and Engineering Research Council, Daresbury, UK) using exact curve wave scattering theory (19Lee P.A. Pendry J.B. Physiol. Rev. 1975; B11: 2795-2811Crossref Scopus (989) Google Scholar, 20Gurman S.J. Binsted N. Ross I. J. Phys. Chem. 1984; 17: 143-151Google Scholar) to analyze the spectra. The energy range was set to 30–650 eV above the edge. Phases were calculated ab initio using Hedin-Lundqvist potentials and von Barth ground states (21Hedin L. Lundqvist S. Solid State Phys. 1969; 23: 1-181Google Scholar). Both single and multiple scattering paths up to 4.5 Å from the metal atom were used to identify and quantify imidazole coordination of histidine ligands by using implemented small molecule data base of the program. After eliminating all non-imidazole atoms of the His unit, the complete imidazole ring was simulated by iterating the distance and Debye-Waller factor of the pivotal (directly coordinating) N atom and the angle φ of the second imidazole N atom for slight distance corrections of the constrained outer shell imidazole atoms. Debye-Waller factors of the outer shell atoms of imidazole rings were constrained assuming the Debye-Waller factors of atoms with similar distance to the absorber to be equal. Since the constraints for the Debye-Waller factors are unique for each parameter set, details are summarized in Tables II and III.Table IIResults of the theoretical EXAFS data analysisfor BcII Open table in a new tab Table IIIResults of the theoretical EXAFS data analysis for CphAView Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab The fitting process included additional constraints for the following parameters. Two coordination clusters were introduced, each having an integer number of ligands. The fit then determined the fractional occupancy of each cluster for the mononuclear enzyme if the metal ions were distributed between the two metal sites. Theoretical simulations were generated by adding shells of scatterers around the central cadmium atoms and iterating the number of scatterers, bond lengths, and Debye-Waller factors in each shell. Additionally, the Fermi energy Ef (edge position relative to calculated vacuum position) was refined to achieve the best fit to the experimental data. The improvement of the fit after the addition of each shell beyond the first was assessed by comparing the residual R-factor (22Joyner R.W. Martin K.J. Meehan P. J. Phys. C Solid State Phys. 1987; 20: 4005-4012Crossref Scopus (293) Google Scholar). 111mCd was produced by the Cyclotron Department at the University Hospital in Copenhagen, Denmark. Preparation and purification of 111mCd is described in Ref. 23Hemmingsen L. Bauer R. Bjerrum M.J. Adolph H.W. Zeppezauer M. Cedergren-Zeppezauer E. Eur. J. Biochem. 1996; 241: 546-551Crossref PubMed Scopus (24) Google Scholar. The PAC spectrometer is described in Ref. 24Bauer R. Danielsen E. Hemmingsen L. Sorensen M.V. Ulstrup J. Friis E.P. Auld D.S. Bjerrum M.J. Biochemistry. 1997; 36: 11514-11524Crossref PubMed Scopus (15) Google Scholar and references therein. In the case of identical, static, and randomly oriented molecules, the perturbation function G2(t) is shown in Equation 1, G2(t)=a0+a1cos(ω1t)+a2cos(ω2t)+a3cos(ω3t)(Eq. 1) with ω1, ω2, and ω3 as the three difference frequencies between the three sublevels of the spin 52 state of the cadmium nucleus (25Bauer R. Q. Rev. Biophys. 1985; 18: 1-64Crossref PubMed Scopus (51) Google Scholar). Note that ω1 + ω2 = ω3. Thus, the Fourier transform of G2(t) exhibits three frequencies for each nuclear quadrupole interaction (NQI). The Fourier transformation was performed as described in Ref. 24Bauer R. Danielsen E. Hemmingsen L. Sorensen M.V. Ulstrup J. Friis E.P. Auld D.S. Bjerrum M.J. Biochemistry. 1997; 36: 11514-11524Crossref PubMed Scopus (15) Google Scholar. The NQI is characterized by the numerically largest diagonal element after diagonalization, chosen as ωzz, which is denoted ω0 and η = (ωxx – ωyy)/ωzz. The relation between these two parameters and the frequencies in G2(t) can be found in Ref. 23Hemmingsen L. Bauer R. Bjerrum M.J. Adolph H.W. Zeppezauer M. Cedergren-Zeppezauer E. Eur. J. Biochem. 1996; 241: 546-551Crossref PubMed Scopus (24) Google Scholar. Thus, from the time dependence of G2(t), ω0 and η, determined through least squares fitting, reflect the coordination geometry of the cadmium ion. In the liquid state, the NQI is time-dependent because of the Brownian reorientation of the protein, described by the rotational diffusion time τR. This has the consequence that G2(t) converges to 0 as a function of time, representing thermal equilibrium and isotropy in the angular correlation between the two γ-rays. The perturbation function A2G2(t), where A2 is the amplitude, was analyzed by a conventional non-linear least squares fitting routine. Satisfactory fitting was obtained with a relative Gaussian distribution δ = Δω0/ω0 applied to all the three frequencies. Non-zero values for δ indicate that the 111mCd nuclei are located in a distribution of surroundings. An NQI is then described by the parameters ω0, η, δ, and τR. In cases where more than a single NQI is present, the perturbation function is the sum of the different perturbation functions, where each NQI is weighted by its population (23Hemmingsen L. Bauer R. Bjerrum M.J. Adolph H.W. Zeppezauer M. Cedergren-Zeppezauer E. Eur. J. Biochem. 1996; 241: 546-551Crossref PubMed Scopus (24) Google Scholar). We have studied the interaction of metallo-β-lactamases with the two diastereomers of captopril, which proved to be competitive inhibitors of both BcII and CphA. Inhibition constants of d- and l-captopril for Me1- and Me2-BcII were determined with imipenem as the substrate (Table I). Since it was not possible to yield reasonable results for the putative binuclear species of CphA, only results for the mononuclear species are presented.Table IEnzymatic activities with imipenem and inhibition constants of d- and l-captopril for Zn(II) and CD(II)-substituted BcII and CphAOrganismMetalsKMkcatkcat/KMKiD-CaptoprilKiL-CaptoprilμMs-1s-1 μM-1μMμMBCIIZn(II)173 ± 10127 ± 71.7444 ± 561 ± 5BCIIZn(II)2330 ± 30276 ± 200.8445 ± 565 ± 5BCIICd(II)1820 ± 393.3 ± 0.80.0040.5 ± 0.11.5 ± 0.2BCIICd(II)2888 ± 750.6 ± 0.030.0006818 ± 125 ± 3CphAZn(II)1100aData published in Ref. 27.625aData published in Ref. 27.6.2572 ± 6950 ± 80CphACd(II)135.5aData published in Ref. 27.0.54aData published in Ref. 27.0.0152.7 ± 0.419 ± 2a Data published in Ref. 27Hernandez V.M. Kiefer M. Heinz U. Soto R.P. Meyer-Klaucke W. Nolting H.F. Zeppezauer M. Galleni M. Frere J.M. Rossolini G.M. Amicosante G. Adolph H.W. FEBS Lett. 2000; 467: 221-225Crossref PubMed Scopus (50) Google Scholar. Open table in a new tab We have investigated the influence of inhibitor binding on metal dissociation constants. In the presence of 25 μmd-captopril and 5 μm BcII, a first Cd(II) ion binds with a KD of 1.6 nm as compared with 8.3 nm in absence of d-captopril (18Hemmingsen L. Damblon C. Antony J. Jensen M. Adolph H.W. Wommer S. Roberts G.C. Bauer R. J. Am. Chem. Soc. 2001; 123: 10329-10335Crossref PubMed Scopus (48) Google Scholar). A second cadmium ion is bound with a KD of 50 μm as compared with 5.9 μm in the absence of the inhibitor (18Hemmingsen L. Damblon C. Antony J. Jensen M. Adolph H.W. Wommer S. Roberts G.C. Bauer R. J. Am. Chem. Soc. 2001; 123: 10329-10335Crossref PubMed Scopus (48) Google Scholar). These KD values are macroscopic constants, and in case of the mononuclear enzymes, they do not reflect any binding site assignment (see below). For CphA, the substitution of cadmium for zinc results in a drastic decrease of Km and kcat. Binding of captopril is much stronger to the cadmium species than to the zinc species with a strong preference for d-captopril (Table I). UV-visible Spectroscopy—Binding of Co(II) to the metal-free enzyme at a [Co(II)]/[enzyme] stoichiometry of 0.8 results in the appearance of a ligand-to-metal charge transfer band at 344 nm and bands in the d-d transition region (400–700 nm). In general, the intensity of the ligand-to-metal charge transfer bands is mainly due to Cys (sulfur)-Co(II) interaction, whereas the d-d transitions are caused by the His-Co(II) interaction (17de Seny D. Heinz U. Wommer S. Kiefer M. Meyer-Klaucke W. Galleni M. Frere J.M. Bauer R. Adolph H.W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Increasing the [Co(II)]/[enzyme] ratio above 1 results in a shift of the charge transfer band to 383 nm (Fig. 1) (17de Seny D. Heinz U. Wommer S. Kiefer M. Meyer-Klaucke W. Galleni M. Frere J.M. Bauer R. Adolph H.W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Besides this difference, the d-d regions are almost identical in shape and intensity at low and high stoichiometry of Co(II) relative to the enzyme. A similar H-site occupancy at low and high stoichiometry reflects a strong preference of Co(II) for the H-site in Co(II)1-BcII (17de Seny D. Heinz U. Wommer S. Kiefer M. Meyer-Klaucke W. Galleni M. Frere J.M. Bauer R. Adolph H.W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Binding of d-captopril to Co(II)-BcII leads to changes both in the charge transfer region and in the d-d region, indicating the binding of an additional ligand and likely changes in the coordination geometry of both sites. The difference spectra between inhibitor-bound and free enzyme at low and high stoichiometry are very similar in the charge transfer region and virtually identical in the d-d region, indicating that the modes of binding for d-captopril to Co(II) are also almost identical at both metal stoichiometries (Fig. 1). It has to be emphasized, however, that even under the conditions used for the Co(II)2-BcII experiments (Fig. 1A), the enzyme was not completely available as the Co(II)2 species. Even at the very high concentration of Co(II) used, a fraction of the enzyme still shows the charge transfer band for the mononuclear enzyme at 348 nm, and thus 10–20% of the enzyme still had Co(II) bound only in the DCH site. Thus, a direct quantitative comparison of absorption coefficients of Co(II)1 and Co(II)2 enzyme is difficult and, thus, a quantitative estimate of relative occupancies of both binding sites for the mononuclear enzyme. EXAFS Spectroscopy—For EXAFS spectroscopy on BcII and CphA, ∼0.8 eq of Cd(II)/enzyme were used to minimize contributions from the eventually formed binuclear species. A 3-fold surplus of d-captopril was added to maximize the abundance of the inhibited species. The EXAFS results are given in Tables II and III for BcII and CphA, respectively. The corresponding spectra are shown in Fig. 2. To illustrate the fitting procedure used, Table II shows two alternative models used for simulation of the spectra for uninhibited Cd(II)1-BcII, namely a 1-cluster and a 2-cluster model. Since it is well known that a single cadmium ion is distributed between the two available binding sites (10Paul-Soto R. Zeppezauer M. Adolph H.W. Galleni M. Frere J.M. Carfi A. Dideberg O. Wouters J. Hemmingsen L. Bauer R. Biochemistry. 1999; 38: 16500-16506Crossref PubMed Scopus (33) Google Scholar, 18Hemmingsen L. Damblon C. Antony J. Jensen M. Adolph H.W. Wommer S. Roberts G.C. Bauer R. J. Am. Chem. Soc. 2001; 123: 10329-10335Crossref PubMed Scopus (48) Google Scholar), a typical 1-cluster model necessarily results in an “averaged” ligand sphere. From the coordination numbers resulting for the 1-cluster model, it becomes obvious that neither the 3-His nor the DCH site is fully occupied (NS ∼0.4; NN/imidazole ∼2.02). The fractional occupation of the DCH site (∼40%) leads to a theoretical value of ∼2.2 for NN/imidazole, which is in contradiction to the simulated value of NN/imidazole ∼2.02. The resulting lack of intensity in the theoretical spectrum is compensated by an added broad contribution of oxygen ligands (high NO and very high Debye-Waller factor) in this simple model. If multiple scattering contributions of histidines are not taken into account, the result becomes even more corrupted by not using constrained imidazole ring units since contributions from nitrogen or oxygen ligands are virtually identical (data not shown). Also, second shell contributions were omitted, which is clearly reflected in the lack in intensity at ∼3.2 Å (Fig. 2C) of the theoretical spectrum. Since the amino acid ligand geometry is well known for BcII from x-ray crystallographic data (Protein Data Bank code 1BVT), we could make use of these data by constructing a 2-cluster model. The atomic distribution in the 2 cluster model was accordingly assigned (3-His and DCH site, respectively). Thus, the problem of correlated coordination numbers and Debye-Waller factors could be solved by fixing the ligand coordination numbers according to the structural data derived from the Protein Data Bank file 1BVT. Thus, the only free remaining coordination number in the 2-cluster model is the one of the cadmium ion itself, which determines the fractional occupation of each cluster. Additionally second shell nitrogen/oxygen ligands with a fixed coordination number were introduced to account for back scattering contributions in the ∼3.2-Å range. For the 2-cluster model, an occupation of 70% results for the H-site. Interestingly the ligand specific distances found are in good agreement with the values obtained with the 1-cluster model. All attempts to generate theoretical spectra with 1-cluster models for the d-captopril-inhibited enzyme (with exactly the same method as used for the uninhibited enzyme) failed in the sense that useful data could not be obtained due to mutual dependences of parameters. Although low R-factors could be obtained, Debye-Waller factors and coordination numbers resulted in unrealistic values (data not shown). In the theoretical 2-cluster model for the d-captopril-inhibited enzyme, it proved to be possible to replace the second shell nitrogen/oxygen contributions in the DCH site by introducing a small molecule model of the Asp side chain. Again, all coordination numbers except the coordination number of Cd(II) in both available binding sites were fixed. The resulting distribution of Cd(II) for the inhibited species shows a 60% occupation of the DCH site. The ligand geometry of Cd(II)1-BcII is roughly similar to th" @default.
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