Oxidation of histidine residues in copper-zinc superoxide dismutase by bicarbonate-stimulated peroxidase and thiol oxidase activities: pulse EPR and NMR studies

In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. 1D NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and Cu(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H(2)O(2)) or cysteine in the absence of bicarbonate anion (HCO(3)(-)), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(II) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO(3)(-) protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 ? away from the Cu(II) site, were completely abolished during both HCO(3)(-)-stimulated peroxidase activity and thiol oxidase activity in the presence of HCO(3)(-) . Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO(3)(-) revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO(3)(-), these modifications were absent. HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities.     

Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy.

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.     

Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases.

The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.     

Investigation of the structure of the blue copper protein from Rhus vernicifera stellacyanin by 1H nuclear magnetic resonance spectroscopy.

The 270-MHz 1H nuclear magnetic resonance spectra of Cu(II), Cu(I), and apo-stellacyanin are reported and compared. The data indicate that little conformational change occurs on reduction of the protein or on removing the copper ion. In the aromatic region of the spectra of the holoprotein, resonances associated with two freely titrating histidines are observed. Two additional sharp resonances are observed in the spectra of the apostellacyanin which are tentatively assigned to additional histidines. This result requires that not more than two histidines can be ligands since there are only four histidines in the whole protein. The absence of methionine has been reported and is one of the possible causes for the difference between stellacyanin and the other copper blue proteins. A comparison of these data with those available for other blue copper proteins, in conjunction with the sequence information, leads to a proposed structure for the copper site in stellacyanin.     

Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus beta-lactamase II: A perturbed angular correlation of gamma-rays spectroscopic study

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.     

Ultraviolet difference spectroscopy of bovine carbonic anhydrase substituted with various divalent metals

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5–9 are reported. For Zn(II) at all pH's and Co(II) at pH ? 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280–292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.     

pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.     

牛红细胞超氧化物歧化酶活性中心的紫外光谱研究

应用差示分光光度法研究了牛红细胞Ｃｕ２Ｚｎ２ＳＯＤ的紫外光谱,归属和讨论了酶活性中心金属离子与配体间全部电荷转移谱带,给出了相应的配体轨道光学电负性,特别研究了涉及Ｚｎ２＋的电荷转移谱带．     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.     

A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning

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 N-terminal 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.     

The oxidation of 3-hydroxyanthranilic acid by Cu,Zn superoxide dismutase: mechanism and possible consequences

Cu,Zn SOD, but not Mn SOD, catalyzes the oxidation of 3-hydroxyanthranilic acid (3-HA) under aerobic conditions. In the absence of O2, the Cu(II) of the enzyme is reduced by 3-HA. One plausible mechanism involves the reduction of the active site Cu(II) to Cu(I), which is then reoxidized by the O2- generated by autoxidation of the anthranilyl or other radicals on the pathway to cinnabarinate. We may call this the superoxide reductase, or SOR, mechanism. Another possibility invokes direct reoxidation of the active site Cu(I) by the anthranilyl, or other organic radicals, or by the peroxyl radicals generated by addition of O2 to these organic radicals. Such oxidations catalyzed by Cu,Zn SOD could account for the deleterious effects of the mutant Cu,Zn SODs associated with familial amyotrophic lateral sclerosis and of the overproduction or overadministration of wild-type Cu,Zn SOD.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

Plastocyanin binding to photosystem I as a function of the charge state of the metal ion: effect of metal site conformation.

The binding of Ag- and Cd-substituted plastocyanin to reduced photosystem 1 of spinach has been studied through the rotational correlation time of plastocyanin measured by the technique of perturbed angular correlation of gamma-rays (PAC). Ag and Cd are used as models for native Cu(I) and Cu(II), respectively. A dissociation constant of 5 microM was found for Ag-plastocyanin, whereas the dissociation constant was at least 24 times higher for Cd-plastocyanin. PAC was further used to characterize the structure of the metal site of Cd- and Ag-plastocyanin. The Cd spectra are characteristic of a planar configuration of one cysteine and two histidines. However, the spectra show an unusual peak broadening and a high degree of internal motion, interpreted as motion of one of the histidines within the plane. (111)Ag decays to (111)Cd, followed by the emission of two gamma-rays used for the PAC experiment. The (111)Ag PAC spectra indicate that one of the coordinating histidines has a different position in the Ag protein than in the Cd protein but that the decay of Ag to Cd causes a relaxation of the position of this histidine to the position in the Cd protein within 20 ns. Binding of Ag-plastocyanin to photosystem I stabilized the Ag metal site structure so that no relaxation was observed on a time scale of 100 ns. This stabilization of the Ag structure upon binding indicates that the metal site structure is involved in regulating how the dissociation constant for plastocyanin depends on the charge of the metal ion.     

Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions

In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakers' yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p β-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.     

Thermal stability and redox properties of M. tuberculosis CuSOD

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule. is the water molecule really necessary for the enzymatic mechanism?

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies.     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.     

Electron paramagnetic resonance measurements of the ferrous mononuclear site of phthalate dioxygenase substituted with alternate divalent metal ions: direct evidence for ligation of two histidines in the copper(II)-reconstituted protein.

The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites. A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofactor, and a mononuclear iron site is the putative site of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin domain, transfers electrons from NADH to the Rieske center. Any of the metal ions, Fe(II), Cu(II), Co(II), Mn(II), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effecting hydroxylation. Nevertheless, studies of how these metal ions affect both the EPR spectra of the reduced Rieske site and the kinetics of electron transfer in the PDO system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples in which iron of the mononuclear site was replaced with Cu(II). The use of (63)Cu(II), in combination with PDO obtained from cultures grown on media enriched in (15)N [using ((15)NH(4))(2)SO(4) as a sole nitrogen source], [delta,epsilon-(15)N]histidine, as well as natural abundance sources of nitrogen, enabled detailed spectral analysis of the superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines are coordinated to the mononuclear site. Coupled with previous studies [Bertini, I., Luchinat, C., Mincione, G., Parigi, G., Gassner G. T., and Ballou, D. P. (1996) J. Bioinorg. Chem. 1, 468-475] that show the presence of one or two water molecules coordinated to the iron, it is suggested that the mononuclear site is similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, for which crystal structures are available. The lack of observable EPR interaction signals between Cu(II) in the mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 A apart, which is similar to that found in the naphthalene dioxygenase crystal structure.