Spectroscopic Characterization of Recombinant Cu,Zn Superoxide Dismutase from <i>Photobacterium leiognathi</i> Expressed in <i>Escherichia coli</i>:  Evidence for a Novel Catalytic Copper Binding Site

Foti, Diego; Curto, B. Lo; Cuzzocrea, G; Stroppolo, Maria Elena; Polizio, Francesca; Venanzi, Mariano; Desideri, Alessandro

doi:10.1021/bi963020f

Cited by 30 publications

(20 citation statements)

References 36 publications

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“…The high pH sensitivity of the monomeric enzyme is confirmed by a titration of the EPR spectrum as a function of pH, showing that the protein-bound copper assumes a biuret type of conformation at much lower pH values (10.5 versus 12.5) than the Cu,Zn-SODs from ox (26) and P. leiognathi (27). These findings, together with the previously reported inactivation in native polyacrylamide gel electrophoresis at pH 8.8 (13,14), clearly demonstrate that the enzyme from E. coli is more sensitive to alkaline denaturation than the highly homologous dimeric protein from the bacterium P. leiognathi.…”

Section: Discussionmentioning

confidence: 87%

“…It did not change in the 5.5-10 pH range, but at pH 10.5 a copper-biuret-type EPR spectrum appeared, suggesting a regular square-planar coordination of the copper ion, as it is bound by four peptide nitrogens in a denatured protein (26). However, this pH-induced modification of the EPR spectrum, which occurs at much lower pH values with respect to other Cu,Zn-SODs (26,27), is completely reversible by lowering the pH to neutrality, at variance with the cases of alkaline denaturation of other copper proteins. Such a change of the EPR spectrum was paralleled by alteration of the enzyme activity, which at pH 10.5 was below the detection limit of the assay method used and was fully recovered by adjusting the solution pH to neutral values.…”

Section: Recovery Of Sod Activity After Dsc Scans and Effect Of Incubmentioning

confidence: 84%

“…The lack of dimeric structure in the E. coli enzyme, which is characterized by a highly polar molecular surface (9), could increase flexibility of this loop and explain its unusual active-site accessibility. Alterations in the active-site structure of the monomeric enzyme may be inferred by the EPR and optical spectra that are clearly different with respect to that of the eukaryotic and P. leiognathi enzymes (26,27). All these observations suggest that the copper environment of E. coli Cu,Zn-SOD may be more susceptible to solvent modifications than that of the dimeric enzymes.…”

Section: Discussionmentioning

confidence: 97%

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Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase

Battistoni

Folcarelli

Cervoni³

et al. 1998

Journal of Biological Chemistry

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To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated. Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66°C at pH 7.8 and at 79.3°C at pH 6.0. The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources. Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea. These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the ␤-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its activesite region.

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Section: Discussionmentioning

confidence: 87%

Section: Recovery Of Sod Activity After Dsc Scans and Effect Of Incubmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 97%

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Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase

Battistoni

Folcarelli

Cervoni³

et al. 1998

Journal of Biological Chemistry

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“…This increases the attractive potential near the SSloop (which is present to a lesser extent in the other SODs, as can be seen from the sign conservation map). It should also be noted that the magnitude of the positive potential in the active site is greater for the P. leiognathi enzyme than for the bovine enzyme, although its rate is identical (25). Although mutations have not yet been reported of charged residues in the SSloop, several studies of mutations of the charged residues in the 7,8 loop (Asp-130, Glu-131, Lys-134) and the nearby Lys-120 (not conserved in prokaryotic SODs) and Arg-141 have been made.…”

Section: Electrostatic Steeringmentioning

confidence: 97%

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

Wade

Gabdoulline

Lüdemann

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

205

171

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To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also inf luence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-inf luenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and ␤-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as ''ionic tethering.'' We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

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“…In this context the x-ray structure of different bacterial Cu,Zn-SODs have been solved (13)(14)(15)(16)(17), and the spectroscopic and catalytic properties of some of these enzymes have been investigated (17)(18)(19)(20)(21). These studies have shown that prokaryotic and eukaryotic Cu,Zn-SODs derive from a common ancestor gene and share a similar three-dimensional fold, based on a flattened Greek-key eight-stranded ␤-barrel and a similar organization of the redox center.…”

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confidence: 99%

A Histidine-rich Metal Binding Domain at the N Terminus of Cu,Zn-Superoxide Dismutases from Pathogenic Bacteria

Battistoni¹,

Pacello²,

Mazzetti³

et al. 2001

Journal of Biological Chemistry

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A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the Nterminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

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Spectroscopic Characterization of Recombinant Cu,Zn Superoxide Dismutase from Photobacterium leiognathi Expressed in Escherichia coli: Evidence for a Novel Catalytic Copper Binding Site

Cited by 30 publications

References 36 publications

Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase

Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

A Histidine-rich Metal Binding Domain at the N Terminus of Cu,Zn-Superoxide Dismutases from Pathogenic Bacteria

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