Perturbed angular correlation spectroscopy and its application to metal sites in proteins: possibilities and limitations

Bauer, Rogert

doi:10.1017/s0033583500004972

Cited by 61 publications

(60 citation statements)

References 78 publications

(108 reference statements)

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“…5) with 1 , 2 , and 3 as the three difference frequencies between the three sublevels of the spin 5/2 state of the cadmium nucleus (21). Note that 1 ϩ 2 ϭ 3 .…”

Section: Site-directed Mutagenesis Production and Purification Of Ementioning

confidence: 99%

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Seny¹,

Heinz²,

Wommer³

et al. 2001

Journal of Biological Chemistry

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117

174

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One high affinity (nM) and one low affinity (M) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-␤-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of ␥-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono-and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.Two zinc binding sites in close proximity are conserved in all metallo-␤-lactamases studied so far. Only two of the metal ion ligands undergo variations between the three different subclasses of the enzyme family (1). The enzyme from Bacillus cereus 569/H/9 (BcII) 1 represents a member of subclass B1 with 3 His ligands in one site and 1 Asp, 1 Cys, and 1 His ligand in the other site (3H 1 and DCH 1 sites, respectively). Various crystal structures of BcII are available, representing mononuclear (2) and binuclear species (3, 4). It was shown earlier that both mono-and binuclear zinc enzymes from B. cereus (5) and Bacteroides fragilis (6) are catalytically active.Although catalytic mechanisms for the enzyme with either one or two zinc ions bound have been discussed (for review see Ref. 7) the respective roles of the two binding sites during catalysis are still unclear. Generally the 3H site is considered to be the primary catalytic site. However, the importance of the DCH site for catalysis became obvious from studies of the C168A mutant. When only one zinc ion is bound to this mutant, it shows a very low activity compared with the wild type, whereas wild type-like activity is almost restored when a second metal ion is bound (5).Perturbed angular correlation (PAC) of ␥-ray spectroscopy provides information on the metal ion coordination geometry through measurement of the nuclear quadrupole interaction (NQI) between the nuclear electric quadrupole moment and the electric field gradient from the surrounding charge distribution. With this method it was possible to demonstrate that the Cd(II) ions in the mononuclear wild type BcII are distributed between the two m...

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“…5) with 1 , 2 , and 3 as the three difference frequencies between the three sublevels of the spin 5/2 state of the cadmium nucleus (21). Note that 1 ϩ 2 ϭ 3 .…”

Section: Site-directed Mutagenesis Production and Purification Of Ementioning

confidence: 99%

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Seny¹,

Heinz²,

Wommer³

et al. 2001

Journal of Biological Chemistry

Self Cite

117

174

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“…1) with 1 , 2 , and 3 as the three difference frequencies between the three sublevels of the spin 5 ⁄2 state of the cadmium nucleus (25). Note that 1 ϩ 2 ϭ 3 .…”

Section: Perturbed Angular Correlation Of ␥-Rays Spectroscopymentioning

confidence: 99%

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

Heinz

Bauer²,

Wommer

et al. 2003

Journal of Biological Chemistry

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D-and L-captopril are competitive inhibitors of metallo-␤-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 ␥-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-␤-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.Metallo-␤-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of ␤-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-␤-lactamases. For all metallo-␤-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-␤-lactamase (BcII), 1 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 (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4), 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 (5). 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 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.The structural investigation of D-and L-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an ...

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“…Changes in the first coordination sphere of the active-site metal were also observed upon binding of coenzyme to the active-site cadmium-substituted LADH studied by "Td-NMR (Bobsein and Myers, 1981) and by PAC (Anderson et al, 1982;Hemmingsen et al, PAC is a method that gives information about types and positions of ligands and is extremely sensitive to changes in the first coordination sphere, for which a movement of a ligand of just a few degrees is detectable. A thorough discussion of PAC theory can be found in Frauenfelder and Steffen (1965) and the applications of PAC to studies of metallo-proteins is described in Bauer (1985). Cd-substituted LADH, which must be used in the PAC experiments, is an active enzyme (Zeppezauer, 1986), binds coenzyme and metal-bound ligands and undergoes conformational changes similar to native Zn-containing LADH (Schneider et al, 1985).…”

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confidence: 99%

The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes

Hemmingsen

Bauer

Bjerrum

et al. 1996

European Journal of Biochemistry

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The coordination geometry of the metal at the active site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and C1-, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD' and decanoic acid and NAD' and Cl-, by using the method of perturbed angular correlation of y-rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X-ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution.The spectral parameters, oo and v, of the experiments, except for the complexes with imidazole, fall into two groups: one with low oo and one with high o, (7 is relatively constant in all experiments). In this work it is clarified that the low o,, values are connected with the presence of a negatively charged solvent ligand. Using an angular-overlap approach to interpret the results, the low cc),, values are found to be compatible with a coordination geometry where the S-Cd-S (Cys174 and Cys46 coordinate to the metal) angle is about 110" as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum, M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren-Zeppezauer, E. (1995) Biochemistry 34, 7145571531, whereas high coo values are compatible with an S-Cd-S angle of 130". The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is believed that alcohols coordinate to the metal as alcoholate ions this could be important for catalysis.Keywords: perturbed angular correlation ; protein conformation ; metal-site structure; function ; flexibility.Horse liver alcohol dehydrogenase (LADH), a dimeric Zndependent enzyme, catalyses the reversible oxidation of primary and secondary alcohols using NAD' as coenzyme. A TheorellChance model is assumed for the catalytic process (Kvassman

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Perturbed angular correlation spectroscopy and its application to metal sites in proteins: possibilities and limitations

Cited by 61 publications

References 78 publications

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes

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