Dina Cocco

Carbamoylation of Cu,Zn-superoxide dismutase by cyanate: Role of lysines in the enzyme action

Reaction with cyanate leads to a reversible change of the EPR spectrum of Cu,Zn‐superoxide dismutase and to time‐dependent carbamoylation of the lysine residues of the enzyme, producing a stable covalent derivative with more negative charge. The carbamoylated enzyme is less active than the native enzyme in spite of unaltered EPR spectra. The extent of this inactivation is much less when the enzyme activity is measured at low ionic strength. These results show that integrity of the active site is not the sole factor playing a role in the enzyme mechanism and that the ionic strength effect is related to electrostatic interactions between O− 2 and surface charges of the protein.

A study of the pH dependence of the activity of porcine Cu,Zn superoxide dismutase

Abstract The enzyme activity of porcine Cu,Zn superoxide dismutase, which has an unusually high isoelectric point, decreases almost linearly with increasing pH between pH 7.5 and 12.0, while EPR and NMR parameters of the copper are titrated only above pH 9.5. Elimination of lysine charges by succinylation abolishes the pH dependence of activity between pH 7.5 and 9.5 and produces identical pH-activity curves for both bovine and porcine enzymes. The pH dependence of activity of the succinylated proteins overlaps that of the spectroscopic parameters of the copper in both enzymes. These results indicate that lysines play a critical role in the rate determining step of the mechanism of the Cu,Zn superoxide dismutases.

19F relaxation as a probe of the oxidation state of Cu, Zn superoxide dismutase. Studies of the enzyme in steady-state turnover

Summary 19F nmr relaxation proved to be a proper method to evaluate the Cu2+/Cu+ ratio at the active site of Cu, Zn superoxide dismutase in either equilibrium or turnover conditions. In the steady-state under fluxes of O2−, the enzyme was found to contain 50% Cu2+, in accord with the equal rates of copper catalytic reduction and oxidation. Previous results giving 75% Cu at the steady-state without change of the overall catalytic constant were confirmed for samples subjected to freeze-drying or freezing-thawing.

Reversible uncoupling of the copper and cobalt spin systems in cobalt bovine superoxide dismutase at low pH

A great deal of spectroscopic evidence, obtained with the Co(II) derivative of bovine superoxide dismutase [1,2], has indicated that the copper and the zinc sites are very close to each other. In particular, EPR studies on the Co(II)-Cu(II) protein have shown that the copper and cobalt spin systems are coupled antiferromagnetically at the temperature used to observe the EPR, and that the magnitude of the coupling is such as the two metal sites have a common ligand, probably an imidazole nucleus [3]. Recent X-ray work [4] has confirmed these suggestions, indicating the imidazole nucleus of His 61 as the bridging group. The present report will show that acid titration of Co(II)-Cu(II) bovine superoxide dismutase modifies the optical spectrum of the cobalt much more than that of the copper and brings about a reversible breaking of the magnetic coupling between the two metal sites. The results are interpreted in terms of protonation of the bridging imidazolate on the cobalt-facing nitrogen.

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.

Aromatic side chains in CU-ZN superoxide dismutases

Abstract 1. 1. The role of aromatic amino acids in two Superoxide dismutases from bovine and human blood was studied. The former contains two tyrosines per mole, whose fluorescence is influenced by the presence of copper in the holoprotein or reconstituted protein. 2. 2. The human Superoxide dismutase contains two tryptophans and no tyrosines. 3. 3. The oxidation of these residues by chemical or photodynamic attack does not bring any significant change in Superoxide dismutase activity. 4. 4. This observation is in keeping with previous work on horse liver Superoxide dismutase which lacks both tyrosine and tryptophans while it shows a fairly high activity.

Reduced anion binding and anion inhibition in Cu, Zn superoxide dismutase chemically modified at lysines without alteration of the rhombic distortion of the copper site

The spectroscopic binding constant (visible absorption and EPR spectra) and the catalytic inhibition constant of N-3 and CN- were measured for bovine Cu, Zn superoxide dismutase chemically modified at all lysines by either succinylation or carbamoylation. These modifications partially inactivate the enzyme (10% and 50% residual activity respectively) but leave the native rhombic geometry of the copper site unaffected. It could thus be shown that the observed reduction of anion affinity of the lysines-modified proteins is related to the decreased positive charge of the protein.

A room temperature magnetic susceptibility study on the cobalt derivatives of cuprozinc superoxide dismutase

Abstract A room temperature magnetic susceptibility study was carried out on several derivatives of Cu, Zn Superoxide dismutase: (1) E, Co(II) in which Co(II) binds to the normal Zn(II) site and the Cu(II) site is empty; (2) Co(II), Zn(II) in which Co(II) binds to the site normally occupied by Cu(II); (3) Co(II), Co(II) in which both the Zn(II) and the Cu(II) are replaced by cobalt; (4) Cu(II), Co(II) in which only the Zn(II) is replaced by cobalt. In this latter derivative the effect of the addition of increasing amounts of SCN − on the magnetic susceptibility was also tested. For all the samples the magnetic susceptibility values resulted to be the sum of the contributions of the single paramagnetic ions and indicate that any magnetic coupling between the two metals is well below kT at room temperature.

Electrostatic interactions in Cu, Zn superoxide dismutase: Effects of Ca(II) and of anions not binding to the copper

Cu,Zn superoxide dismutase (EC 1.15.1.1) from bovine erythrocyte was found to enhance Tb(III) luminescence in a way suggestive of the presence of specific sites binding calcium. Binding of Ca(II) had no effect on the enzyme activity. However, the low-temperature EPR spectra of the enzyme-bound copper were modified into a more axial line shape by Ca(II) and Tb(III), but not by other cations. This effect was not observed by room-temperature EPR and was interpreted as being due to a long-range conformational effect on the copper-binding site occurring on freezing when specific charged amino acid side-chains are neutralized. This interpretation is supported by similar effects observed with anions that neither have inhibitory effect on the activity nor bind to the copper. These results indicate the presence of specific electrostatic interactions of the protein moiety of Cu,Zn superoxide dismutase and the occurrence of changes of the symmetry of the copper site when the protein is frozen under certain conditions.

The binding of copper ions to copper-free bovine superoxide dismutase. Applied aspects

The catalytic activity, k = 3 × 109 M−1 [1], close to the diffusion limit and the very high relaxivity of 19H− nucleus, RF ∼ 107 M−1 sec−1 [2], due to the copper in the native site of CuZn superoxide dismutase, make possible the measurement of very low concentrations of these enzymes. In connection with the high sensitivity in the enzyme detection the reconstitution of the native site from Cu++ and the copper -free enzyme besides to offer a good insight into the interaction copper-protein [3–5] presents some interesting applied aspects. The measurement of copper concentrations much lower than ppb is a good example of these applications. For this purpose the polarographic [6] and the pulsed NMR techniques [2] appear very suitable since they can sense superoxide concentrations of the order of 10−11 and 10−8 M respectively. Using these techniques, and owing to the very high affinity of the copper ion for its native site, the binding of Cu++ to the copper-free enzyme has been easily followed in a variety of experimental conditions. In particular the addition of known amounts of CU++ to the copper depleted superoxide dismutase, at [Cu++]/[protein] ⩽ 0.1, causes a linear and reproducible increase of both the activity and the relaxivity after few minutes incubation. The method results to be highly specific for the Cu++ and allows, as measured by the polarographic activity and by 19F relaxivity, to detect the ion with sensitivity of the order of 10−10 and 2 × 10−8 M respectively.