Alessandro Desideri

Crystal structure of yeast Cu,Zn superoxide dismutase: Crystallographic refinement at 2.5 Å resolution☆

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis

Several point mutations in the gene coding for human Cu,Zn superoxide dismutase have been reported as being responsible for familial amyotrophic lateral sclerosis (FALS). However, no direct demonstration has been provided for a correlation between total superoxide dismutase activity and severity of the FALS pathology. In order to get a better insight into the mechanism (s) underlying the FALS phenotype, we have investigated the activity and the copper binding properties of the single mutant H46R, which is associated with a Japanese form of FALS. We have shown that this mutant is structurally stable but lacks significant enzyme activity and has impaired capability of binding catalytic copper. The mutant protein can be fully reconsituted with copper in vitro but its ESR spectrum displays an axial shape quite different from that of the wild‐type.

Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase pH-DEPENDENT, REVERSIBLE DENATURATION OF THE MONOMERIC ENZYME FROM ESCHERICHIA COLI

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 theE. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66u2009°C at pH 7.8 and at 79.3u2009°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 theE. 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 β-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of theE. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region.

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site : evidence for Co-ordinated mutation of charged amino acid residues

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

The essential dynamics of Cu, Zn superoxide dismutase : Suggestion of intersubunit communication

A 300-ps molecular dynamics simulation of the whole Cu, Zn superoxide dismutase dimer has been carried out in water, and the trajectory has been analyzed by the essential dynamics method. The results indicate that the motion is defined by few preferred directions identified by the first four to six eigenvectors and that the motion of the two monomers at each instant is not symmetrical. The vectors symmetrical to the eigenvectors are significantly sampled, suggesting that, on average, the motions of the two subunits will exchange. Large intra- and intersubunit motions involving different subdomains of the protein are observed. A mechanical coupling between the two subunits is also suggested, because displacements of the loops surrounding the active site in one monomer are correlated with the motion of parts of the second toward the intersubunit interface.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a spectroscopic and thermodynamic study.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated quantitatively by EPR and absorbance spectroscopy. The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and bakers yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins. Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x 10(-6) M and < or = 5 x 10(-8) M, respectively, at pH 7.0 and 10 degrees C. The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-1, at 10 degrees C).

Crystal structure solution and refinement of the semisynthetic cobalt-substituted bovine erythrocyte superoxide dismutase at 2.0 Å resolution☆

The semisynthetic Co-substituted bovine erythrocyte superoxide dismutase (SOD) has been crystallized in a new crystalline form and the structure determined at 2.0 A (1 A = 0.1 nm) resolution. The crystals belong to space group P2(1)2(1)2(1) with cell constants: a = 51.0, b = 147.6, c = 47.5 A, and contain one dimeric molecule of 32,000 M(r) per asymmetric unit. The structure has been solved by molecular replacement techniques using the Cu,Zn bovine enzyme as a search model, and refined by molecular dynamics with the crystallographic pseudo-energy term, followed by conventional crystallographic refinement. The R-factor for the 18,964 unique reflections in the resolution range from 10.0 to 2.0 A is 0.176 for a model comprising 2188 protein atoms and 200 solvent molecules; the root-mean-square deviation from the ideal bond lengths is 0.010 A, and the average atomic temperature factor is 26.5 A2. The dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. The subunit has as its structural scaffolding the conventional SOD-flattened antiparallel eight-stranded beta-barrel, with three external loops. The co-ordination geometry of the metal center in the active site is fairly well preserved when compared with the native Cu,Zn bovine enzyme. Co2+ is in tetrahedral co-ordination, while the Cu2+ ligands show an uneven distortion from the square planar geometry. The least-squares superposition of the metals ligands and the catalytically important Arg141 of the native and Co-substituted enzyme yields a root-mean-square value of 0.401 A, the largest deviation occurring at the Co2+ ligand Asp81. An additional copper ligand, compatible with a water molecule, is observed at 2.38 A from Cu2+ in the active-site channel, at the supposed binding site of the O2- anion substrate. Several ordered water molecules have been observed on the protein surface and in the active-site channel; their structural locations coincide remarkably with those of related water molecules found in the crystal structure of the phylogenetically distant superoxide dismutase from yeast.

Three-dimensional structure of Xenopus laevis Cu,Zn superoxide dismutase b determined by X-ray crystallography at 1.5 A resolution.

Xenopus laevis Cu,Zn superoxide dismutase (recombinant isoenzyme b) has been crystallized and the structure determined at 1.49 A resolution. The crystals belong to space group P2(1)2(1)2(1), with cell constants a = 73.33, b = 68.86, c = 59.73 A, and contain one dimeric molecule of M(r) 32 000 per asymmetric unit. The structure was solved by molecular-replacement techniques using the semisynthetic Cu,Co bovine enzyme as search model, and refined by molecular dynamics with a crystallographic pseudo-energy term. During the final steps, positional and anisotropic thermal parameters of the atoms were refined. The R factor for the 49 209 unique reflections in the 10.0-1.49 A resolution range is 0.104, for a model comprising 2023 protein atoms, two Cu(2+), two Zn(2+), and 353 water molecules. The overall temperature factor for the model, including solvent, is 20.3 A(2), while the calculated r.m.s. coordinate error for the refined model is 0.036 A. As suggested by the primary structure homology to any other known intracellular eukaryotic superoxide dismutase (> 50%), the typical structural scaffolding of flattened antiparallel eight-stranded (beta-barrel is well conserved in X. laevis Cu,Zn superoxide dismutase b, together with the coordination geometry of the metal centers in the active site. The higher thermal stability of the bb X. laevis superoxide dismutase homodimer, with respect to dimers involving the a-type isoenzyme subunit(s), can be related, on the basis of the high-resolution structure, to side-chain and solvent interactions centered on residue Tyr149, in both b-type subunits. The analysis of the overall solvent structure reveals a number of equivalent water molecule sites in the two subunits, and in homologous superoxide dismutase models. Their locations are discussed in detail and classified on the basis of their structural role.

Azide, cyanide, fluoride, imidazole and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative: A comparative study

Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c.

Crystal structure of the cyanide-inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K

The crystal structure of cyanide‐inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R‐factor for the 27,299 reflections in the 10.0‐1.7 Å resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 Å in subunit A, and 0.27 Å in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.