Cyanide binding to Cu, Zn superoxide dismutase. An NMR study of the Cu(II), Co(II) derivative

The Cu,Co superoxide dismutase derivative, in which the native Zn(II) was replaced by Co(II), was investigated by 1H NMR spectroscopy at pH 7.0 in the presence of CN- and N-3. Addition of either anion produced large but remarkably different variations in the position of the histidine proton signals bound to the metal cluster. The resonances of the histidines bound to the copper broadened at low CN- concentrations (6 X10(-5)-16.5 X 10(-3) M KCN, in the presence of 1.5 mM protein) and narrowed again, with changed chemical shifts at [KCN] greater than 10(-2) M. At 7 degrees C two resonances split into two pairs of lines as a function of [CN-]. The temperature dependence of these resonances, in the presence of nonsaturating [CN-], suggests a slow exchange between two forms of the protein-bound copper in the presence of the anion. The apparent activation parameters associated with the interconversion of the two species indicate a local conformational change in the presence of CN-. No evidence of temperature dependence was seen in the spectrum in the presence of N-3, which, on the other hand, was fully removed from the copper by addition of CN-. No evidence was obtained for removal by CN- of a histidine bound to the copper as previously reported for low affinity anions at pH 5.5 (Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monanni, R., and Scozzafava, A. (1985) J. Am. Chem. Soc. 107, 4391-4396). These results indicate that CN- has a unique pattern of binding to the enzyme copper. Since catalytic and structural data indicate that CN- is the only appropriate substrate analogue for the Cu,Zn superoxide dismutase, data from anions with much less affinity may lead to misleading conclusions on the mechanism of anion and substrate binding to the enzyme.     

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Cobalt bovine superoxide dismutase. Reactivity of the cobalt chromophore in the copper-containing and in the copper-free enzyme.

1. The reactivity of the zinc site of bovine superoxide dismutase has been probed by observing optical and electron paramagnetic resonance changes, under several conditions, of the Co(II)-substituted protein. 2. Only in the absence of copper are the optical and electron paramagnetic resonance spectra of the cobalt chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the copper-containing protein appears to involve the water molecule bound to the copper and does not affect the magnetic coupling between copper and cobalt. 3. The reaction of cyanide with the copper-free Co(II) protein leads to a slow detachment of cobalt from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(II) carbonic anhydrase (Haffner, P. H. and Coleman, J. E. (1975) J. Biol. Chem. 250, 996--1005.) 4. Acid titration modifies the Co(II) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(II)--Cu(II) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. 5. H2O2 modifies the cobalt chromophore only in the presence of copper. Magnetic coupling between Cu(II) and Co(II) seems to be still present after H2O2 inactivation of the enzyme.     

Increase of Cu,Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein.

Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.     

X-ray absorption investigation of a unique protein domain able to bind both copper(I) and copper(II) at adjacent sites of the N-terminus of Haemophilus ducreyi Cu,Zn superoxide dismutase

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.     

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.     

X-ray absorption spectroscopy of the copper chaperone HAH1 reveals a linear two-coordinate Cu(I) center capable of adduct formation with exogenous thiols and phosphines

The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm. The implications of these findings for mechanisms of copper transport are discussed.     

Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear Overhauser enhancement of the isotropically shifted 1H-NMR lines of the Cu,Co derivative

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu,Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X-ray crystallography.     

An unexpected formation of the spectroscopic Cu(I)-semiquinone radical by xenon-induced self-catalysis of a copper quinoprotein

Plant copper/quinone amine oxidases are homodimeric enzymes containing Cu(II) and a quinone derivative of a tyrosyl residue (2,4,5-trihydroxyphenylalanine, TPQ) as cofactors. These enzymes catalyze the oxidative deamination of primary amines by a classical ping-pong mechanism, i.e. two distinct half-reactions, enzyme reduction by substrate followed by its re-oxidation by molecular oxygen. In the first half-reaction two forms of the reduced TPQ have been observed, the colorless Cu(II)-aminoquinol and the yellow Cu(I)-semiquinolamine radical so that this enzyme may be referred to as a "protein-radical enzyme". The interaction of xenon, in aqueous solutions, with the copper/TPQ amine oxidase from lentil (Lens esculenta) seedlings has been investigated by NMR and optical spectroscopy. NMR data indicate that xenon binds to the protein. Under 10 atm gaseous xenon and in the absence of substrates more than 60% native enzyme is converted into Cu(I)-semiquinolamine radical species, showing for the first time that both monomers in the dimer can generate the radical. Under the same experimental conditions the copper-free lentil enzyme is able to generate an intermediate absorbing at about 360 nm, which is assigned to the product Schiff base quinolaldimine which, to the best of our knowledge, has never been observed during the catalytic mechanism of plant amine oxidases. A possible role of the lysine residue responsible for the formation of Cu(I)-semiquinolamine and quinolaldimine, is proposed.     

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.     

Binding of polyaminocarboxylate chelators to the active-site copper inhibits the GSNO-reductase activity but not the superoxide dismutase activity of Cu,Zn-superoxide dismutase

In addition to its superoxide dismutase (SOD) activity, Cu,Zn-superoxide dismutase (CuZnSOD) catalyzes the reductive decomposition of S-nitroso-L-glutathione (GSNO) in the presence of thiols such as L-glutathione (GSH). The GSNO-reductase activity but not the superoxide dismutase (SOD) activity of CuZnSOD is inhibited by the commonly used polyaminocarboxylate metal ion chelators, EDTA and DTPA. The basis for this selective inhibition is systematically investigated here. Incubation with EDTA or DTPA caused a time-dependent decrease in the 680 nm d-d absorption of Cu(II)ZnSOD but no loss in SOD activity or in the level of metal loading of the enzyme as determined by ICP-MS. The chelators also protected the SOD activity against inhibition by the arginine-specific reagent, phenylglyoxal. Measurements of both the time course of SNO absorption decay at 333 nm and oxymyoglobin scavenging of the NO that is released confirmed that the chelators inhibit CuZnSOD catalysis of GSNO reductive decomposition by GSH. The decreased GSNO-reductase activity is correlated with decreased rates of Cu(II)ZnSOD reduction by GSH in the presence of the chelators as monitored spectrophotometrically at 680 nm. The aggregate data suggest binding of the chelators to CuZnSOD, which was detected by isothermal titration calorimetry (ITC). Dissociation constants of 0.08 +/- 0.02 and 8.3 +/- 0.2 microM were calculated from the ITC thermograms for the binding of a single EDTA and DTPA, respectively, to the CuZnSOD homodimer. No association was detected under the same conditions with the metal-free enzyme (EESOD). Thus, EDTA and DTPA must bind to the solvent-exposed active-site copper of one subunit without removing the metal. This induces a conformational change at the second active site that inhibits the GSNO-reductase but not the SOD activity of the enzyme.     

Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase

The reaction of the copper of (Cu,Zn)-superoxide dismutase with diethyldithiocarbamate was studied at pH = 7.4 and the results obtained led to a reaction scheme basically different from the conclusion of a previous study (Misra, H. P. (1979) J. Biol. Chem. 254, 11623-11628). The analysis of optical and ESR spectra at 9 and 35 GHz, at different ligand/protein ratios and reaction times, showed that a ternary diethyldithiocarbamate. Cu(II).protein complex never formed in spectroscopically detectable amounts. The system is described in any condition as the mixture, in variable proportions, of only two components, that is the diethyldithiocarbamate-free (Cu(II) chelate and the copper-depleted protein. The formation of a catalytically active copper-diethyldithiocarbamate intermediate with distinct optical and ESR spectra was also ruled out by kinetic studies, which demonstrated that enzyme inactivation strictly parallels the binding of diethyldithiocarbamate as monitored by optical absorption and ESR. Separation of the copper complex from the protein was obtained for the first time, and the procedure was suitable for rapid preparation of reconstitutable copper-free superoxide dismutase.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

Oxygen-independent induction of enzyme activities related to oxygen metabolism in yeast by copper

Aerobic growth of Saccharomyces cerevisiae in the presence of CuSO4 (between 0.1 and 1 mM) caused a generalized induction of major enzyme activities involved in 'housekeeping' routes of oxygen metabolism (cytochrome oxidase, glutathione peroxidases and catalase) which were comparable to or higher than that observed with Cu,Zn-superoxide dismutase. Fumarase and glutathione transferase, tested as controls for oxygen-unrelated activities, were found to decrease under the same conditions. In the absence of oxygen, copper addition to yeast resulted in significant increases of Cu,Zn-superoxide dismutase and glutathione peroxidases and a slight increase of cytochrome oxidase, with catalase remaining undetectable irrespective of whether or not copper was present. Other metal ions tested (Mn2+, Co2+) were unable to produce such effects. It is concluded that copper has a general inducing effect on enzymes related to metabolism of oxygen and oxygen derivatives, which is mediated neither by formation of O2-. and H2O2 nor by interaction with copper-specific apoproteins. These results point to a general role of copper as regulator of the expression of major enzyme activities involved in biological oxygen activation.     

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.     

Preparation of selectively metal-free and metal-substituted derivatives by reaction of Cu--Zn superoxide dismutase with diethyldithiocarbamate.

Incubation of Cu--Zn superoxide dismutase with diethyldithiocarbamate at increasing ligand/protein ratios and subsequent high-speed centrifugation led to proportional removal of copper from the protein, at variance with previous results [Misra (1979) J. Biol. Chem. 254, 11623--11628]. No zinc was lost, even at very high excesses of chelating agent. In this way a copper-free protein could be readily prepared, with avoidance of the critical pH condition and the dialysis step required in a previous method employing cyanide. The holoprotein was fully reconstituted from the copper-free protein by stoicheiometric re-addition of copper. From the mixture of metal-depleted forms originated by treatment with slight diethyldithiocarbamate excess, the protein containing copper only on one subunit, [Cu1--Zn2], could be isolated by preparative column electrophoresis. This species reproducibly showed 25% more specific activity (catalytic constant per copper) than that of the native or reconstituted [Cu2--Zn2] protein. This may result from long-range conformational effects between the active sites. By adding Co2+ ions to the vacant copper site of [Cu1--Zn2] a hybrid molecule containing Cu(II) on one subunit and Co(II) in the homologous site of the other subunit was prepared. Its activity, referred to copper, was identical with that of the native protein.     

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.     

Interaction of Cu(II)with His-Val-Gly-Asp and of Zn(II) with His-Val-His,Two Peptides at the Active Site of Cu,Zn-Superoxide Dismutase

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). We have already studied the interaction of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II). As a continuation of this work and for comparison purposes we have also studied the interaction of Zn(II) with His-Val-His and Cu(II) with His-Val-Gly-Asp using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. Histamine type of coordination is observed for/ZnAH/²⁺, /ZnA/⁺, /ZnA²H/⁺ and/ZnA²/ in acidic pH while deprotonation of coordinated water molecules is observed at higher pH. /CUB/ species is characterized by the formation of a macrochelate and histamine type coordination. Its stability results in the suppression of amide deprotonation which occurs at high pH resulting in the formation of the highly distorted from square planar geometry 4N complex/CuBH_-3/³.