Roberto Monnanni

2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum

Oxidized ferredoxin from Clostridium pastetirianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the β‐CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys β‐CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding α‐CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.

Protease susceptibility of zinc - and APO-carboxypeptidase A

Proteases in preparations of carboxypeptidase A progressively inactivate solutions of the apoenzyme but not the metal-containing enzyme. Free amino acids generated by proteolysis interfere with spectral studies after reconstituting the apoenzyme with cobalt. Purification by affinity chromatography eliminates this effect. Affinity-purified apoenzyme is susceptible to digestion with chymotrypsin but the metalloenzyme is not.

13 C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A

Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.

13C NMR studies of D- and L-phenylalanine binding to cobalt (II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.

The pH dependent properties of metallotransferrins: a comparative study

The dependence on pH of the absorption and circular dichroic spectra of iron(III), cobalt(III) and copper(II) transferrins has been (re)investigated. In the alkaline region, the CD profiles of iron(III) and cobalt(III) transferrin are essentially pH independent up to pH 11; only for very high pH values (pH > 11) is breakdown of the cobalt(III) and iron(III) transferrin derivatives observed, without evidence of conformational rearrangements. By contrast, the CD profiles of copper transferrin show drastic changes in shape around pH 10; these spectral changes, which are fitted to a pKa of ~10.4, are interpreted in terms of a substantial rearrangement of the local environment of the copper ions at high pH. Although the CD spectra of copper transferrin at alkaline pH strictly resemble those observed upon addition of modifier anions, the mechanism of site destabilization in the two cases is different; at variance with the case of modifier anions, our results suggest that the high pH form of copper transferrin still contains the synergistic anion. A13C NMR experiment has confirmed this view. In the acidic region, iron(III) and cobalt(III) transferrins are stable down to pH ~6. For lower pH values progressive metal detachment is observed without evidence of conformational changes; around pH 4.5 most bound metals are released. In the case of the less stable copper-transferrin, metal removal from the specific binding sites is already complete around pH 6.0; in concomitance with release from the primary sites, binding of copper ions to secondary sites is observed. Additional information has been gained from CD experiments in the far UV. The pH dependent properties of iron(III), cobalt(III) and copper(II) transferrin are discussed in the frame of the present knowledge of transferrin chemistry, particular emphasis being attributed to the comparison between tripositive and bipositive metal derivatives.

Nickel carbonic anhydrase: a re-examination of the electronic spectra with the help of CD spectra

Abstract The electronic and CD spectra of nickel(II)-substituted bovine carbonic anhydrase, and of its adduct with p -toluenesulfonamide, have been re-recorded in the rangers 8–30 X 10 3 cm −1 and 10–30 X 10 3 cm −1 , respectively. Although the positions of the main absorption are consistent with six-coordination of the metal ion in the enzyme active site, their relative intensity and the detection of other transitions suggest the operativity of strong low-symmetry components. Therefore five-coordinated rather than octahedral chromophores have been considered in order to account for the observed spectroscopic properties.

Different behavior of sulfonamides with respect to copper-substituted bovine and human carbonic anhydrases

Abstract Spectroscopic evidences for different types of derivatives, between sulfonamides and copper-substituted carbonic anhydrases are reported. Depending on the type of sulfonamide and on the particular isoenzyme used, the ligands substitute the coordinated water molecule or bind the metal at a different site without removing the water. A four-coordinated pseudotetrahedral structure is proposed in the former case and a five-coordinated one in the latter.

The dependence of cis-diamminedichloroplatinum(II) binding to DNA upon the GC content: a thermal and spectrophotometrical investigation

Abstract The binding of cis -diamminedichloroplatinum(II) ( cis -DDP) to DNA is investigated as a function of the GC content and of the cis -DDP concentration. Analysis of the melting curves and of the UV and CD spectra shows that binding of cis -DDP to DNA is highly sequence specific, GC rich DNAs being destabilized more effectively. At low levels of platination, stabilization of GC- and destabilization of AT-base pairs can be noted. In AT rich DNA, cis - DDP binding favours the B→ A conformational transition. It is also shown that, in addition to specific interactions between cis -DDP and DNA, several non-specific processes take place.

Investigation of cobalt(II) substituted carboxypeptidase a interacting with azide and cyanate ions

Abstract Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3 − and NCO − , but not NCS − , in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating k cat and K M . The electronic and 1 H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.

Azide and chloride binding to carboxypeptidase A in the presence of L-phenylalanine

The interaction of chloride with native and cobalt (Co)-substituted carboxypeptidase-A (CPD) has been investigated by 35Cl nuclear magnetic resonance (NMR) spectroscopy in the presence and absence of L-Phe. The affinity constants of azide and chloride toward the Co(II)CPD·L-Phe complex have been measured by electronic spectroscopy. The correlation times determining T1 and T2 for the 35Cl nuclei are related to movements inside the cavity. In the presence of L-Phe, the anions bind to the metal with a relatively high affinity at pH values below 6. Anion binding to the Co enzyme can be analyzed in terms of the three protonation state model for the enzyme (EH2 α EH α E). In the presence of L-Phe, Cl− binds to EH2 but not to EH or E while N3− binds ∼ 34 times tighter to EH2 than EH, while binding to E is undetectable. The pH-binding constant profile for N3− binding to the Co(II)CPD·L-Phe complex is characterized by pKa values of 5.3 and 7.6. The lower value corresponds to the pKa of the group that characterizes the pH-kcat profile for Co(II)CPD while the higher pKa likely reflects the group that characterizes the pH-pKm profile in the alkaline pH region. This group has a pKa of 8.9 in the Co and native enzymes but is shifted to 7.6 in the presence of L-Phe. The present results in conjunction with prior spectral and kinetic studies suggest azide preferentially binds to the enzyme when L-Phe has its amino group protonated. The highly synergistic binding between L-Phe to the active site and azide to the metal may be viewed as caused by replacement of the Glu-270 metal-water hydrogen-bonding network by an electrostatic interaction between ionized Glu-270 and the protonated amino group of L-Phe. Thus, the formation of this E·I species allows ready displacement of the metal bound water by azide.