Structure and dynamics of copper-free SOD: The protein before binding copper.

The solution structure of the copper-free state of a monomeric form of superoxide dismutase (153 amino acids) was determined through (13)C and (15)N labeling. The protein contained two mutations at the native subunit-subunit interface (F50E and G51E) to obtain a soluble monomeric species and a mutation in the active site channel (E133Q). About 93% of carbon atoms, 95% of nitrogen atoms, and 96% of the protons were assigned. A total of 2467 meaningful NOEs and 170 dihedral angles provided a family of 35 conformers with RMSD values of 0.76 +/- 0.09 A for the backbone and 1.22 +/- 0.13 A for all heavy atoms. The secondary structure elements, connected by loops, produce the typical superoxide dismutase Greek key fold, formed by an eight-stranded beta-barrel. The comparison with the copper-bound monomeric and dimeric structures shows that the metal ligands have a conformation very close to that of the copper-bound forms. This feature indicates that the copper-binding site is preorganized and well ordered also in the absence of the copper ion. The active-site channel shows a sizable increase in width, achieving a suitable conformation to receive the copper ion. The histidines ring NH resonances that bind the copper ion and the region around the active-site channel experience, as found from (15)N relaxation studies, conformational exchange processes. The increased width of the channel and the higher mobility of the histidine rings of the copper site in the copper-free form with respect to the holoprotein is discussed in terms of the process of copper insertion.

Cleavage of the iron-methionine bond in c-type cytochromes: crystal structure of oxidized and reduced cytochrome c(2) from Rhodopseudomonas palustris and its ammonia complex.

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.

1H NMR Study of the Reduced Cytochrome c' from Rhodopseudomonas palustris Containing a High-Spin Iron(II) Heme Moiety.

The assignment of the hyperfine shifted signals of the reduced cytochrome c' from Rhodopseudomonas palustris has been obtained through saturation transfer experiments with assigned signals of the high-spin oxidized protein and through tailored experiments to reveal proton-proton dipolar connectivities in paramagnetic molecules. The peculiar shift pattern consisting of the 1-, 8-, and 5-methyl signals shifted upfield and the 3-methyl signal downfield, which is shared by all cytochromes c' so far described, has been semiquantitatively related to the orientation of the histidine plane with respect to the iron-heme nitrogen axes. The research is meaningful with respect to the use of paramagnetic NMR as a tool to obtain direct structural information on all high spin iron(II) heme containing systems, including deoxyglobins.

From an inactive prokaryotic SOD homologue to an active protein through site-directed mutagenesis.

It is known that several prokaryotic protein sequences, characterized by high homology with the eukaryotic Cu,ZnSODs, lack some of the metal ligands. In the present work, we have stepwise reintroduced the two missing copper ligands in the SOD-like protein of Bacillus subtilis, through site-directed mutagenesis. The mutant with three out of the four His that bind copper is not active, whereas the fully reconstituted mutant displays an activity of about 10% that of human Cu,ZnSOD. The mutated proteins have been characterized in solution and in the solid state. In solution, the proteins experience conformational disorder, which is believed to be partly responsible for the decreased enzymatic activity and sheds light on the tendency of several human SOD mutants to introduce mobility in the protein frame. In the crystal, on the contrary, the protein has a well-defined conformation, giving rise to dimers through the coordination of an exogenous zinc ion. The catalytic properties of the double mutant, which might be regarded as a step in an artificial evolution from a nonactive SOD to a fully functioning enzyme, are discussed on the basis of the structural and dynamical properties.

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: from largely unstructured in solution to ordered in the crystal.

Little is known about prokaryotic homologs of Cu,Zn superoxide dismutase (SOD), an enzyme highly conserved among eukaryotic species. In 138 Archaea and Bacteria genomes, 57 of these putative homologs were found, 11 of which lack at least one of the metal ligands. Both the solution and the crystal structures of the SOD-like protein from Bacillus subtilis, lacking two Cu ligands and found to be enzymatically inactive, were determined. In solution, the protein is monomeric. The available nuclear Overhauser effects, together with chemical-shift index values, allowed us to define and to recognize the typical Cu,Zn SOD Greek beta-barrel but with largely unstructured loops (which, therefore, sample a wide range of conformations). On the contrary, in the crystal structure (obtained in the presence of slight excess of Zn), the protein is well structured and organized in covalent dimers held by a symmetric bridge consisting of a Zn ion bound to an Asp-His dyad in a tetrahedral geometry. Couples of dimers held by hydrophobic interactions and H bonds are further organized in long chains. The order/disorder transition is discussed in terms of metal binding and physical state.

Solution structure of Apo Cu,Zn superoxide dismutase: role of metal ions in protein folding.

The solution structure of the demetalated copper, zinc superoxide dismutase is obtained for the monomeric Glu133Gln/Phe50Glu/Gly51Glu mutant through NMR spectroscopy. The demetalated protein still has a well-defined tertiary structure; however, two beta-strands containing two copper ligands (His46 and His48, beta4) and one zinc ligand (Asp83, beta5) are shortened, and the sheet formed by these strands and strands beta7 and beta8 moves away from the other strands of the beta-barrel to form an open clam with respect to a closed conformation in the holoprotein. Furthermore, loop IV which contains three zinc ligands (His63, His71, and His80) and loop VII which contributes to the definition of the active cavity channel are severely disordered, and experience extensive mobility as it results from thorough (15)N relaxation measurements. These structural and mobility data, if compared with those of the copper-depleted protein and holoprotein, point out the role of each metal ion in the protein folding, leading to the final tertiary structure of the holoprotein, and provide hints for the mechanisms of metal delivery by metal chaperones.

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization.

The solution structure of homodimeric Cu2Zn2 superoxide dismutase (SOD) of 306 aminoacids was determined on a 13C, 15N and 70% 2H labeled sample. Two-thousand eight-hundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a family of 30 conformers with an rmsd from the average of 0.78 +/- 0.11 and 1.15 +/- 0.09 A for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop to 0.65 +/- 0.09 and 1.08 +/- 0.11 A for the backbone and heavy atoms, respectively. The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural differences between the two subunits as well as between different molecules in the unit cell. The elements of secondary structure, i.e. eight beta sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the contrary, loops IV and VI show some disorder. The residues of the active cavity are well defined whereas within the various subunits of the X-ray structure some are disordered or display different orientation in different X-ray structure determinations. The copper(I) ion and its ligands are well defined. This structure thus represents a well defined model in solution relevant for structure-function analysis of the protein. The comparison between the solution structure of monomeric mutants and the present structure shows that the subunit-subunit interactions increase the order in loop II. This has the consequences of inducing the structural and dynamic properties that are optimal for the enzymatic function of the wild-type enzyme. The regions 37-43 and 89-95, constituting loops III and V and the initial part of the beta barrel and showing several mutations in familial amyotrophis lateral sclerosis (FALS)-related proteins have a quite extensive network of H-bonds that may account for their low mobility. Finally, the conformation of the key Arg143 residue is compared to that in the other dimeric and monomeric structures as well as in the recently reported structure of the CCS-superoxide dismutase (SOD) complex.

The evolutionarily conserved trimeric structure of CutA1 proteins suggests a role in signal transduction.

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit beta-sheet formation. Each subunit consists of a ferredoxin-like (beta1alpha1beta2beta3alpha2beta4) fold with an additional strand (beta5), a C-terminal helix (alpha3), and an unusual extended beta-hairpin involving strands beta2 and beta3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.

Solution structure and characterization of the heme chaperone CcmE.

The covalent attachment of the heme cofactor in c-type cytochromes is a surprisingly complex process, which in bacteria involves a number of different proteins. Among the latter, the ccmE gene product is known to perform a key role in the heme delivery pathway in Gram-negative bacteria. The solution structure of the soluble domain of apo-CcmE from Shewanella putrefaciens was determined through NMR spectroscopy on a 13C,15N-labeled sample. The structure is characterized by a compact core with large regions of beta structure, while the N-terminal and C-terminal regions are essentially unstructured. The overall folding is similar to that of the so-called oligo-binding proteins (OB fold). Solvent-exposed aromatic residues, conserved in all CcmE homologues, have been found in the proximity of His131, the putative heme-binding residue, that could have a role in the interaction with heme. No interaction between CcmE and heme, as well as between CcmE and holocytochrome c, could be detected in vitro by electronic spectroscopy or by NMR. The data available suggest that the heme transfer process is likely to involve a heterooligomeric protein complex and occur under a tight enzymatic control.

Protein stability and mutations in the axial methionine loop of a minimal cytochrome c.

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.