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.     

The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited.

The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl2. The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disulfide bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Human SOD1 before harboring the catalytic metal: solution structure of copper-depleted, disulfide-reduced form

SOD1 has to undergo several post-translational modifications before reaching its mature form. The protein requires insertion of zinc and copper atoms, followed by the formation of a conserved S-S bond between Cys-57 and Cys-146 (human numbering), which makes the protein fully active. In this report an NMR structural investigation of the reduced SH-SH form of thermostable E,Zn-as-SOD1 (E is empty; as is C6A, C111S) is reported, characterizing the protein just before the last step leading to the mature form. The structure is compared with that of the oxidized S-S form as well as with that of the yeast SOD1 complexed with its copper chaperone, CCS. Local conformational rearrangements upon disulfide bridge reduction are localized in the region near Cys-57 that is completely exposed to the solvent in the present structure, at variance with the oxidized forms. There is a local disorder around Cys-57 that may serve for protein-protein recognition and may possibly be involved in intermolecular S-S bonds in familial amyotrophic lateral sclerosis-related SOD1 mutants. The structure allows us to further discuss the copper loading mechanism in SOD1.     

Molecular cloning and characterization of copper/zinc-superoxide dismutase of Paragonimus westermani

Superoxide dismutases (SODs; EC 1.15.1.1) play important roles in the protection of the parasites against cellular oxygen-mediated killing of the hosts. A copper/zinc-containing SOD (Cu/Zn-SOD) was identified previously from lung fluke, Paragonimus westermani. To expand our understanding of P. westermani SOD, we isolated a complementary DNA encoding a Cu/Zn-SOD, expressed the active enzyme in Escherichia coli, and characterized its biochemical properties. The deduced amino acid (aa) sequence of the gene shared up to 73.7% identities with Cu/Zn-SODs of other helminths and shared well-conserved characteristic motifs and essential aa residues involved in coordinating copper and zinc enzymatic functions. Recombinant Cu/ Zn-SOD exhibited comparable biochemical properties with that of the native enzyme, including pH optima and potassium cyanide-and hydrogen peroxide-sensitive inhibition profiles. The active enzyme consisted of 2 identical subunits covalently linked by disulfide bonds. The enzyme was constitutively expressed throughout various developmental stages of the parasite. The levels increased as P. westermani matured and plateaued in adult stage. Our result suggests the enzyme might play an important role for parasites to survive in the hosts through its superoxide anion-detoxifying function.     

Effect of local environment and protein on the mechanism of action of superoxide dismutase

Quantum mechanical simulations of the mechanism of action of superoxide dismutase (SOD) indicate that the presence of Arg-141 in the active site of the enzyme is responsible for the formation of an intermediate complex between superoxide and the enzyme in which the copper is not reduced. The analysis of the local environmental effects of Arg-141 shows that this residue prevents the reduction of copper by forming a hydrogen bond to superoxide and by generating an electric field in the active site that opposes the transfer of an electron from superoxide to copper. The protein enhances the effect of the opposing field generated by Arg-141. Local changes in the environment of the copper ion, simulated by stretching the Cu-NE2 (His-61) bond also do not induce an electron transfer from superoxide to copper. The protein increases the energies required for this stretch through the electric field it generates near the active site. These results are further support for the new proposed mechanism of action of SOD which is based on the inability of superoxide to reduce the cupric ion in the enzyme.     

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.     

Selection of novel structural zinc sites from a random peptide library

Zinc ion (Zn(2+)) can be coordinated with four or three amino acid residues to stabilize a protein's structure or to form a catalytic active center. We used phage display selection of a dodecamer random peptide library with Zn(2+) to identify structural zinc sites. The binding specificity for Zn(2+) of selected sequences was confirmed using enzyme-linked immunosorbent and competitive inhibition assays. Circular dichroism spectra indicated that the interaction with Zn(2+) induced a change in conformation, which means the peptide acts as a structural zinc site. Furthermore, a search of protein databases revealed that two selected sequences corresponded to parts of natural zinc sites of copper/zinc superoxide dismutase and zinc-containing ferredoxin. We demonstrated that Zn(2+)-binding sequences selected from the random combinatorial library would be candidates for artificial structural zinc sites.     

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.     

Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis

At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two CuII ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-strandedbeta-sheet and one alpha-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2═O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.     

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.     

Crystallographic determination of reduced bovine superoxide dismutase at pH 5.0 and of anion binding to its active site

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C222₁ crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis.     

Differential inactivation of alcohol dehydrogenase isoenzymes in Zymomonas mobilis by oxygen.

Zymomonas mobilis is endowed with two isoenzymes of fermentative alcohol dehydrogenase, a zinc-containing enzyme (ADH I) and an iron-containing enzyme (ADH II). The activity of ADH I remains fully conserved, while ADH II activity decays when anaerobic cultures are shifted to aerobiosis. This differential response depends on the metal present on each isoenzyme, since pure preparations of ADH I are resistant to oxidative inactivation and preparations of zinc-containing ADH II, obtained by incubation of pure ADH II with ZnCl2, showed no modification of the target for oxidative damage (His277-containing peptide). It was consistently found that the activity of the zinc-containing ADH II, once submitted to oxidative treatment, was fully restored when iron was reintroduced into the enzyme structure. These results indicate that zinc bound to these proteins plays an important role in the protection of their active centers against oxidative damage and may have relevant biochemical and physiological consequences in this species.     

Molecular cloning and amino acid sequence of rat enkephalinase

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.     

The red light of the helium-neon laser reactivates superoxide dismutase

The effect of low-energy helium-neon laser (HNL) on enzymatic activity, absorbtion spectra and electron paramagnetic resonance (EPR) signals of superoxide dismutase (SOD) from bovine erythrocytes in acid medium were investigated. It was found that incubation during 2 hours at pH 5.9 led to eventually complete inactivation of the enzyme. The subsequent illumination of inactivated SOD by HNL brought about the enzyme reactivation. Both absorption and EPR-spectra were changed after incubation at pH 5.9. These changes may be attributed to protonation of histidine residue in the enzyme active site. After laser irradiation both absorption and EPR spectra were restored to those typical of native enzyme at pH 8.2. In a model system, copper-histidine complex, absorption maximum was shifted from 632-633 nm at pH 5.8 to 639-640 nm at pH 8.5-9.0. The similar long-wave length shift of the maximum was observed after illumination by HNL at pH 5.8. It may be postulated that the photoreactivation of SOD consists essentially in deprotonation of His-61 residue in the enzyme active site and subsequent recovery of imidazole bridge between copper and zinc which had been destroyed at low pH. Since many other enzymes possess similar copper-histidine structures in their active sites, one may expect diverse effects of red (laser) light on the enzyme activity.     

Trifluoroethanol-induced activity and structural changes in bos taurus copper- and zinc-containing superoxide dismutase

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in organisms exposed to oxygen. Copper- and zinc-containing SOD (Cu/Zn-SOD) catalysis and the change in folding behavior of this enzyme in response to inactivators are therefore of interest. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of a Cu/Zn-SOD from Bos taurus. We found that TFE inactivated the enzyme and disrupted the tertiary and secondary structures of Cu/Zn-SOD. Kinetic studies showed that TFE-induced inactivation of Cu/Zn-SOD follows first-order reaction kinetics and that TFE binding sites are distinct from the copper- and zinc-containing active site. These structural changes occurred prior to enzyme activity loss. A computational docking simulation of Cu/Zn-SOD and TFE (binding energy of Dock 6.3: -11.52 kcal/mol) suggested that THR37, ASP40, and GLU119, which are located near the active site, interact with TFE. Evaluation of the ligand binding kinetics of Cu/Zn-SOD during unfolding in the presence of TFE combined with computational prediction allowed us to gain insight into the inactivation of Cu/Zn-SOD.