Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2005), 14:387-394. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muñoz, I. G.
Right arrow Articles by Montoya, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muñoz, I. G.
Right arrow Articles by Montoya, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The crystal structure of an eukaryotic iron superoxide dismutase suggests intersubunit cooperation during catalysis

Inés G. Muñoz1, Jose F. Moran2, Manuel Becana3 and Guillermo Montoya1

1 Structural Biology and Biocomputing Program, Spanish National Cancer Center (CNIO), Macromolecular Crystallography Group, 28029 Madrid, Spain
2 Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, 31006 Pamplona, Spain
3 Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), 50059 Zaragoza, Spain

Reprint requests to: Guillermo Montoya, Structural Biology and Biocomputing Program, Spanish National Cancer Center (CNIO), Macromolecular Crystallography Group, c/o Melchor Fernández Almagro 3, 28029 Madrid, Spain; e-mail: gmontoya{at}cnio.es; fax: +00-34-912246976.

(RECEIVED July 8, 2004; FINAL REVISION September 30, 2004; ACCEPTED September 30, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Superoxide dismutases (SODs) are a family of metalloenzymes that catalyze the dismutation of superoxide anion radicals into molecular oxygen and hydrogen peroxide. Iron superoxide dismutases (FeSODs) are only expressed in some prokaryotes and plants. A new and highly active FeSOD with an unusual subcellular localization has recently been isolated from the plant Vigna unguiculata (cowpea). This protein functions as a homodimer and, in contrast to the other members of the SOD family, is localized to the cytosol. The crystal structure of the recombinant enzyme has been solved and the model refined to 1.97 Å resolution. The superoxide anion binding site is located in a cleft close to the dimer interface. The coordination geometry of the Fe site is a distorted trigonal bipyramidal arrangement, whose axial ligands are His43 and a solvent molecule, and whose in-plane ligands are His95, Asp195, and His199. A comparison of the structural features of cowpea FeSOD with those of homologous SODs reveals subtle differences in regard to the metal–protein interactions, and confirms the existence of two regions that may control the traffic of substrate and product: one located near the Fe binding site, and another in the dimer interface. The evolutionary conservation of reciprocal interactions of both monomers in neighboring active sites suggests possible subunit cooperation during catalysis.

Keywords: antioxidants; iron superoxide dismutase; manganese superoxide dismutase; X-ray crystallography; protein–protein interaction

Abbreviations: SOD, superoxide dismutase • PDB, Protein Data Bank • RMS, root-mean-square • ROS, reactive oxygen species • Tm, transition temperature • CuZnSOD, copper zinc superoxide dismutase • FeSOD, iron superoxide dismutase • Vu_FeSOD, Vigna unguiculata iron superoxide dismutase • Ec_FeSOD, Escherichia coli iron superoxide dismutase • Po_FeSOD, Pseudomonas ovalis iron superoxide dismutase • Mt_FeSOD, Methanobacterium thermoautotrophicum iron superoxide dismutase • Ss_FeSOD, Sulfolobus solfataricus iron superoxide dismutase • MnSOD, manganese superoxide dismutase • Hu_MnSOD, Homo sapiens manganese superoxide dismutase • Ec_MnSOD, Escherichia coli manganese superoxide dismutase

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04979505.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
A problem shared by all aerobic organisms is that between 1% and 3% of the oxygen consumed by respiration is partially reduced to reactive oxygen species (ROS), such as the superoxide radical, hydrogen peroxide, and the hydroxyl radical. ROS can oxidize DNA, proteins, and lipids, leading ultimately to cell death. Superoxide radicals exhibit only moderate reactivity, but can be the precursors of highly oxidizing hydroxyl radicals. SODs catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide, and thus constitute a major antioxidant defense mechanism. They are present in virtually all aerobes and in several anaerobes. There are three known classes of plant SODs, each coordinating with a different metal cofactor. CuZnSODs are found in the cytosol, chloroplasts, nucleus, and apoplast (Kanematsu and Asada 1991), MnSODs are observed in mitochondria and peroxisomes (Fridovich 1995), and FeS-ODs are present in chloroplasts (Kliebenstein et al. 1998). SODs catalyze a two-step reaction in which superoxide radicals are dismutated into hydrogen peroxide, while a metal cofactor cycles between the reduced and oxidized forms (Halliwell and Gutteridge 1999).

The structures of several SODs of each type have already been reported (six FeSODs, three MnSODs, and seven CuZnSODs). Sequence and structure comparisons show that the MnSOD and FeSOD groups are closely related to each other, whereas the CuZnSODs appear to have evolved independently. Structurally, MnSOD and FeSOD appear to be variants of the same enzyme (Stallings et al. 1984). Both contain an {alpha}/{beta} fold, which differs from the Greek key {beta}-barrel of CuZnSOD (Tainer et al. 1982). MnSOD and FeSOD are typically observed to be homodimers or homotetramers. Each 200-residue monomer is bound to a metal ion. The active sites of these enzymes are specific for their respective metal ions and for the superoxide anion. They exhibit a conserved structure that consists of a group of metal-binding residues enclosed by a shell of residues. Although both enzymes have the ability to bind either Mn or Fe, the replacement of the corresponding metal ion in the native SOD decreases enzyme activity (Ose and Fridovich 1976; Yamakura and Suzuki 1980), a result that is probably due to inappropriate redox potentials (Brock and Harris 1977; Vance and Miller 1998).

In this report we present for the first time the structure of a recombinant FeSOD from the plant Vigna unguiculata (cowpea). This type of protein, when present in an eukaryotic organism, has only been located in the chloroplasts of plants. To our knowledge, this is the first FeSOD that has been clearly observed in the cytosol (Moran et al. 2003). Therefore, contrary to the widely held view, FeSODs in plants are not only restricted to the chloroplasts, but also probably constitute a defensive mechanism against oxidative stress associated with senescence. Our model contributes to a deeper understanding of this family of enzymes, providing structural data for the most evolved member of this important family of proteins. A structural comparison between cowpea FeSOD and other SODs from distinct organisms belonging to different kingdoms confirms the existence of regions, close to the active site, that contain key residues that have essential functions during enzyme catalysis. Furthermore, the crystal structure corroborates the presence of interactions between residues of both monomers that have been conserved over evolutionary time and that most likely play a critical role during enzyme catalysis.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Overall structure
Protein characterization and crystallization have previously been reported (Moran et al. 2003; Muñoz et al. 2003) (see Supplemental Material). The crystal structure of cowpea iron superoxide dismutase (Vu_FeSOD), an iron-containing protein with 238 amino acid residues, was solved by molecular replacement and refined to a crystallographic R-factor of 14.8% and R-free of 19.2% (Table 1Go). The crystal belongs to the monoclinic space group C2 and has one monomer per asymmetric unit (Muñoz et al. 2003) (Table 1Go). Vu_FeSOD overall fold is an {alpha}/{beta} fold (Fig. 1AGo). There was no electron density evident to model the previous first 13 residues of the amino acid sequence so that the model starts at residue Lys14 (Fig. 1AGo). The monomer consists of two structural domains: an N-terminal helical domain composed of two long antiparallel {alpha}-helices, which are separated by a small {alpha}-helix and a C-terminal {alpha}/{beta} domain, which contains a central three-stranded antiparallel {beta}-sheet and four {alpha}-helices (Fig. 1AGo). The first 15 amino acid residues at the N terminus form an extended region that packs antiparallel to helix {alpha}1. This helix is slightly kinked around the conserved Lys46 (Edwards et al. 1998). The helix {alpha}1 is connected by a turn to helix {alpha}2 forming a helical hairpin structure. The residues (Gly–Thr) at the tip of the helical hairpin could not be built into the electron density map. The overall B-factors of the modeled residues at the extremes of this loop (43 Å2 and 47 Å2) are higher than the average B-factor (Table 1Go). The short helix {alpha}2 connects the two long {alpha}1 and {alpha}3 helices. The helix {alpha}2 is also present in the structures of the bacterial FeSOD from Escherichia coli (Ec_FeSOD) and Pseudomonas ovalis (Po_FeSOD). This helix is substituted by a long loop that extends towards the solvent in the structures of the Archaea Metanobacterium thermoautothrophicum (Mt_FeSOD), Sulfolobus solfataricus (Ss_FeSOD), and in the human MnSOD (Hu_MnSOD) (Fig. 1BGo). The C-terminal domain is an {alpha}/{beta}-type fold and consists of a three-stranded antiparallel {beta}-sheet with helices {alpha}4, {alpha}5, {alpha}6, and {alpha}7 on one side. A large loop exposed to the solvent joins strands {beta}1 and {beta}2 (Fig. 1AGo). The majority of this loop is absent in other Fe and MnSODs (Fig. 1BGo). In our model, the loop has a gap comprising nine residues, from Asp155 to Ala165, in which no clear electron density was present to model them. Again, the overall B-factor at the two edges of the gap (49 Å2 and 60 Å2) are above the average value of the structure (Table 1Go). The metal binding site is located at the interface between the N- and C-terminal domains. The helices {alpha}2 and {alpha}3 contribute to the binding site with two residues, His43 and His95, whereas the {beta}3 strand and the subsequent loop supply the other two metal ligands, Asp195 and His199.


View this table:
[in this window]
[in a new window]
  		Table 1. Data collection and refinement statistics
 


View larger version (52K):
[in this window]
[in a new window]
  		Figure 1. (A) C{alpha} ribbon representation of the FeSOD structure of Vigna unguiculata. Helical segments are shown in blue; {beta}-strands, in purple; and loop regions, in gray. Helices are labeled from {alpha}1 to {alpha}7 and {beta}-strands from {beta}-1 to {beta}-3. The catalytic residues are shown in detail, with the solvent molecule in red and the iron atom in green. Residues Lys14 at the N-terminal, Ala238 at the C-terminal, and Val59–Thr62, Asp155–Ala165 corresponding to the residues at the two gaps have been numbered for clarity. (B) Sequence alignment of Hu_MnSOD with FeSOD from plants (Vu_FeSOD), bacteria (Ec_FeSOD and Po_FeSOD), and Archaea (Mt_FeSOD and Ss_FeSOD). All the structures were pairwise aligned based on the three-dimensional structure of FeSOD from Vu_FeSOD. The yellow boxes indicate the residues that form the catalytic site in all the enzymes, magenta boxes indicate the residues that compose the shell opposite to the catalytic site, and green boxes indicate the position of the residues that are involved in the interface. A was prepared using Molscript (Kraulis 1991) and POV-Ray (http://www.povray.org). B was prepared using ALSCRIPT (Barton 1993).

 
The active site geometry
The structure and active site geometry are well defined by the electron density map (Fig. 2Go). Although the position of the metal in the active site and the residues nearby are similar to other SODs, several differences in distances and amino acid positions are noteworthy. The Fe is pentacoordinated by four residues (His43, His95, Asp195, and His199) and a solvent molecule that has been proposed to be a hydroxide ion (Figs. 2Go,3Go; Stallings et al. 1991). The five ligands form a spatially arranged trigonal bipyramide around the iron atom (Stallings et al. 1985). The position of the Fe defines a cavity inside the protein (Fig. 3Go), which is hidden and protected by four additional residues, His47, Tyr51, Gln91, and Trp197. These four residues are involved in control of the transit of substrate and product (Maliekal et al. 2002). Figure 3Go illustrates the position of these residues, which form a secondary shell that covers the entrance to the active site. The three aromatic residues are conserved among FeSODs, with the exception of Ss_FeSOD (Fig. 1BGo), in which a phenylalanine is substituted for a tryptophan. Although the glutamine residue and its side chain conformation, which points to the active site, is conserved in Vu_FeSOD, Po_FeSOD and Ec_FeSOD, a histidine can be found in its place in the structures of the Archaea FeSODs (Mt_FeSOD and Ss_FeSOD) (Fig. 1BGo). The amide group of the histidine side chain occupies a position similar to the glutamine in all FeSOD structures (data not shown). This conserved arrangement suggests that the amide group, which has conserved its position in the active site of different SODs, not only performs an important role during catalysis (Yikilmaz et al. 2002), but may also be involved in metal specificity (Hunter et al. 2002).



View larger version (124K):
[in this window]
[in a new window]
  		Figure 2. FeSOD active site and geometry. View of a 2(|Fo| – |Fc|) omit map at 1.97 Å contoured at 1 {sigma}, showing the residues and the solvent molecule involved in the binding of the catalytic iron. The omit map was calculated with the program OMIT in the CCP4 package. Figures 3Go, 4Go, and 5Go were prepared using O (Jones 1991), and Molray (Harris and Jones 2001).

 


View larger version (61K):
[in this window]
[in a new window]
  		Figure 3. C{alpha} ribbon diagram showing a detailed view of the amino acids that form the active site in Vu_FeSOD. The catalytic residues, His 43, 95, and 199, and Asp 195, are colored in yellow, while the second shell amino acids, His47, Tyr51, Gln91, Trp144, and Trp197, are in magenta. The solvent molecules, which form part of the secondary shell, opposite to the active site, are colored purple. The hydrogen bond network between the shell forming residues is shown as gold bubbled lines.

 
The structure of the Hu_MnSOD active site reveals features similar to those of the Archaea Ss_FeSOD and Mt_FeSOD enzymes (Fig. 1BGo). The main difference is due to substitution by an alanine of the tryptophan in the group of residues surrounding the active site. Even though the glutamine is also present, the side-chain distance to the proposed hydroxide ion is shorter (2.99 Å compared to 3.20 Å in Vu_FeSOD) than in other reported structures, which suggests a stronger interaction. However, its contact with the tyrosine is weaker; there is a distance of more than 4 Å in contrast with 2.90 Å in the case of Vu_FeSOD (Fig. 3Go).

Crystal packing and oligomerization
The FeSOD enzymes can be found in homodimeric or homotetrameric form depending on the organism of origin. Although the protein behaves as a dimer in size exclusion chromatograhy (data not shown), our crystal structure has one protein molecule per asymmetric unit with dimensions 45 x 31 x 36 Å (Muñoz et al. 2003). However, crystal packing reveals how the molecule generates a firm contact with one of the other protein monomers. These two monomers are related by a twofold axis (Fig. 4Go). The packing between the two molecules is tight, indicating that this dimer represents the usual biological oligomerization state of the enzyme, where the active sites are located in close proximity to the dimer interface with a distance of 18.01 Å between the two Fe atoms (Fig. 4Go). A comparison of these two molecules with other dimeric FeSOD structures revealed a similar arrangement of the two monomers (Ringe et al. 1983; Stallings et al. 1983). This, together with the buried surface(1100 Å2) (Nicholls et al. 1991) between the protomers, strongly supports the idea that the dimer represents the biological unit.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 4. Interaction between the FeSOD monomers in the crystal. The depicted dimer is the biological unit in vivo. Each monomer is colored in blue and gold. The distance between iron atoms is 18.01 Å. The residues involved in iron binding are represented in ball and stick, and the iron atom and the water molecule are colored in green and red, respectively.

 
From the interface of the two molecules, the solvent region can be divided in two areas: one between the monomers that generates a channel filled with solvent molecules, and a second around each monomer cavity, where the Fe atom is located. Each cavity leads to the channel giving the appearance of a funnel at each site. Figure 5Go (see Supplemental Material) shows the contacts of all conserved residues involved in the formation of the solvent filled channel. The separation between the two molecules ranges between 2.7 Å and 2.9 Å. A close contact is found at the entrance of the channel, where both monomers interact by hydrogen bonds with their respective Ser142 (2.89 Å) and a water molecule. Another important interaction can be observed between the Glu198–OE2 of each monomer and the His199–ND1 of both catalytic centers (2.73 Å), which involves three additional water molecules. The interaction between Tyr202 from one monomer with His47 from the other (Tyr202–OH to His47–NE2, 2.58 Å) also merits attention. These interactions are conserved in other FeSOD and MnSOD structures, and the hydrogen bond between His47 and Tyr202 seems to be involved in the enzymatic mechanism of Ec_MnSOD (Edwards et al. 2001). The position of the residues and water molecules involved in the channel and cavity arrangement is basically identical in the structures of Vu_FeSOD, Ec_FeSOD, Po_FeSOD, and Ss_FeSOD, all of which form a dimer (Fig. 1BGo). In the case of Mt_FeSOD and Hu_MnSOD, the biological unit is a tetramer arranged as a couple of dimers. In this case, the residues in the active site and surroundings from one monomer conserve the interactions mentioned above with one of the neighboring monomers, but not with the other two. This implies that dimeric interactions have been evolutionarily conserved (Fig. 1BGo), and suggests an active role for the residues of one monomer in the active center of the other during catalysis. Thus, the subunits of the tetrameric FeSODs and MnSODs should perform the enzymatic reaction in a pairwise form.



View larger version (61K):
[in this window]
[in a new window]
  		Figure 5. View of the channel created between the monomers. Backbone representation in blue and gold, respectively. The catalytic residues are colored in yellow; the shell forming residues, in magenta; and the residues of the channel involved in interactions between monomers, in green. The water molecules are colored in pale blue. The interactions between Ser142, Glu198, and Tyr202 of one monomer and Ser142, His199, and His47 of the other monomer are depicted in detail.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
We present the first structure of a eukaryotic FeSOD. This enzyme from cowpea is the first one in its family found in the cytosol (Moran et al. 2003). This implicates Vu_FeSOD in cellular mechanisms of free-radical elimination other than those related to photosynthetically produced superoxide. The structure has been solved to 1.97 Å, which allows us to describe the structural characteristics of this new enzyme and compare them with other reported structures.

A close examination suggests that the simpliest pathway for the superoxide radical to reach the active site is controlled by the Gln91, Tyr51, His47, and Trp197, the residues that build a shell around the metal center (Fig. 3Go; Whittaker and Whittaker 1997). Hence, the superoxide radical should move through the cavity, interact with these residues, and ultimately be guided to the active site where dismutation takes place. The importance of these residues has been highlighted before (Jackson et al. 2002; Maliekal et al. 2002; Yikilmaz et al. 2002). The task of Trp197 is probably essential for the function of Gln91, His47, and Tyr51, since it covers the upper part of the cavity entrance and thus provides an appropriate environment for catalysis. Trp144, which is located near the active site, is also found in all the FeSOD and MnSOD structures (Figs. 1BGo, 3Go). The positioning of both tryptophans implies little room for alternative conformations at the active site (Fig. 3Go). The shape and properties of the other FeSODs and MnSODs are identical around most of this group of residues on the side facing the binding cleft. Therefore, although subtle differences in the mechanisms have been reported (Jackson et al. 2002), the driving of the susbtrate probably occurs in a manner common to all of them.

However, it is still not clear how protons are delivered to the substrate in the second step of the enzymatic reaction. The possible existence of several pathways to the active site has been considered before (Edwards et al. 2001). One hypothetical candidate for this role is Tyr51, which may be involved in the direct release and transfer of protons from water molecules found outside the active pocket (Lah et al. 1995). A second candidate may be the conserved Tyr202 from the neighboring monomer. Both the water molecules and the residues themselves, which are properly positioned to deliver protons to the substrate via these tyrosines, are conserved among most of the known SOD structures. As a result, this hypothesis would imply the requirement of a dimer to supply the conserved Tyr202 involved in the enzymatic reaction.

How the oligomerization state of the enzyme relates to the catalytic mechanism is a question that has not been fully answered yet. We can confirm that the residues located in the surroundings of the channel are similar in all the FeSOD and MnSOD structures, and that these residues build a similar network of interactions (Figs. 1BGo, 5Go). The water molecules at the monomers interface are most likely an important factor for the supply of protons to both catalytic sites. Furthermore, important contacts between the subunits in the dimer have been conserved (Ringe et al. 1983; Stallings et al. 1983; Edwards et al. 2001). A view of the channel indicates a crucial role for several interactions (Fig. 5Go). As mentioned before, Tyr202 from one monomer might participate in the catalysis of the neighboring active site through its interaction with His47. This histidine is located in the shell of residues around the Fe binding site (Fig. 3Go). The disruption of this interaction reduces the superoxide dismutase activity to 30% to 40% in Ec_MnSOD (Edwards et al. 2001). Another contact of one monomer with the active site of the other involves a hydrogen bond between Glu198 and His199 (Fig. 5Go). This interaction is of utmost importance to preserve the dimer formation in Ec_MnSOD (Edwards et al. 1998).

The conservation of these residues from Archaea to Eukarya and their interactions argue in favor of their important role in enzyme stability and function (Fig. 1BGo). In addition, a close analysis of the oligomerization state of the different SODs reveals not only a similar organization among the dimeric SODs (superpositions: Vu_FeSOD-Po_FeSOD 0.862 Å RMSD for 370 C{alpha}; Vu_FeSOD-Ec_FeSOD 0.827 Å RMSD for 372 C{alpha}; and Vu_FeSOD-Ss_FeSOD 1.18 Å RMSD for 332 C{alpha}), but also the arrangement of the tetrameric SODs as a dimer of dimers (Vu_FeSOD-Mt_FeSOD, 1.18 Å RMSD for 349 C{alpha}; and Vu_FeSOD-Hu_MnSOD, 1.22 Å RMSD for 351 C{alpha}). In the case of the tetrameric arrangement, the key interactions are conserved in a pairwise form, including not only the distances among the key residues and the water molecules in the channel, but also the distance between the metal active sites, which is around 18 Å in all of them.

Considering the absence of monomeric SODs in nature together with all the previous information, there is a strong indication that the dimer is the minimal catalytically active form of this type of enzyme. This would imply the possibility of intersubunit cooperation during catalysis. Therefore, based on the analysis of the reported structures, the tetrameric SODs should function as a couple of dimers that undergo catalysis in an independent manner.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Protein purification, crystallization, and data collection
Preparation and characterization of Vu_FeSOD have previously been described (Moran et al. 2003; see Supplemental Material). Crystallization experiments and data collection have been reported by Muñoz et al. (2003). Images were processed and scaled with the HKL program (Otwinowski and Minor 1997) and programs of the CCP4 package (Collaborative Computational Project 1994). Statistics for the crystallographic data are summarized in Table 1Go.

Structure solution
The structure was solved using the molecular replacement method as implemented in the program EPMR (Kissinger et al. 1999). The search model was based on an alignment of the amino acid sequence of the Vu_FeSOD with that of Po_FeSOD, obtained using the program CLUSTALW (Higgins et al. 1994), and finally, it was built by modification of the model of Po_FeSOD found in the Protein Data Bank (Benson et al. 2000) (entry 1DT0). The correct solution was the highest peak in both rotation and translation searches including data between 15.0 Å and 4.0 Å resolution, with a final correlation coefficient of 0.522. A 2Fo - Fc map showed clear and contiguous electron density for the peptide backbone and for many of the side-chains of the protein.

Model building, refinement, and analysis of the final model
Five percent of the reflections of the data set were set aside for free R-factor calculations during refinement (Brünger 1992). Positions where the sequence differed were designated alanine and glycine. The electron density map was calculated using only the working set of reflections, and the model was rebuilt where the electron density supported changes. When clear density was observed in place of the side chain expected in Vu_FeSOD, the model was mutated accordingly and the side chain fitted into the density. The resulting model was then refined against the 1.97 Å data set using CNS (Brünger et al. 1998) for the first round of the refinement, including a rigid body minimization followed by simulated annealing (Cartesian starting at 5000 K). The R-factor and R-free values after this first cycle were 0.331 and 0.346, respectively. Further rounds of model mutation/rebuilding were performed using the program O (Jones et al. 1991). Refinement proceeded with the program REFMAC5 (Murshudov et al. 1999) including a rigid-body refinement as the first step. The data were anisotropic, and the most successful refinement strategy made use of Babinet’s bulk solvent correction (Moews and Kretsinger 1975), combined with overall anisotropic scaling and individual anisotropic temperature factor refinement using maximum likelihood as implemented in REFMAC5. Several rounds of rebuilding using the program O and the placement of the iron atom and the water molecules into the electron density, resulted in the final model. The statistics after crystallographic refinement of this model are summarized in Table 1Go. All the structure superpositions were performed with the use of the program O lsq routine (Jones et al. 1991). Coordinates and the corresponding structure factor data, have been deposited in the RCSB Protein Data Bank (Benson et al. 2000) with entry code 1unf.


   Footnotes
 
Supplemental material: see www.proteinscience.org


   Acknowledgments
 
I.G.M. thanks CNIO for a postdoctoral fellowship. J.F.M. thanks CSIC-EU for an I3P postdoctoral contract and MCyT for a Ramón y Cajal contract. Full financial support was obtained through a CNIO internal grant to G.M., and partial support by grant PB98-0522 (DGICYT) to M.B. We thank F. Blanco, T. Zimmerman, and T. Williams for careful reading of the manuscript.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Barton, G.J. 1993. ALSCRIPT: A tool to format multiple sequence alignments. Protein Eng. 6: 37–40.[Free Full Text]

Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Rapp, B.A., and Wheeler, D.L. 2000. GenBank. Nucleic Acids Res. 16: 10881–10890.

Brock, C.J. and Harris, J.I. 1977. Superoxide dismutase from Bacillus stearothermophilus: Reversible removal of manganese and its replacement by other metals. Biochem. Soc. Trans 5: 1537–1539.[Medline]

Brünger, A.T. 1992. Free R value: A novel statistical quantity for assessing the accuracy of crystal structures. Nature 355: 472–475.[CrossRef]

Brünger, A.T., Adams, P.D., Clore, G.M. DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography and NMR system (CNS): A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54: 905–921.[CrossRef][Medline]

Collaborative Computational Project. 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763.[CrossRef][Medline]

Edwards, R.A., Baker, H.M., Whittaker, M.M., Whittaker, J.W., Jameson, G.B., and Baker, E.N. 1998. Crystal structure of Escherichia coli manganese superoxidase dismutase at 2.1 Å resolution. JBIC 3: 161–171.

Edwards, R.A., Whittaker, M.M., Whittaker, J.W., Baker, E.N., and Jameson, G.B. 2001. Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity. Biochemistry 40: 4622–4632.[CrossRef][Medline]

Fridovich, I. 1995. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64: 97–112.[CrossRef][Medline]

Halliwell, B. and Gutteridge, J.M.C. 1999. Free radicals in biology and medicine, 3rd ed. Oxford University Press, Oxford.

Harris, M. and Jones, T.A. 2001. Molray—A web interface between O and the POV-Ray ray tracer. Acta Crystallogr. D Biol. Crystallogr. 57: 1201–1203.[CrossRef][Medline]

Higgins, D., Thompson, J., and Gibson, T.J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680.[Abstract/Free Full Text]

Hunter, T., Bannister, J.V., and Hunter, G.J. 2002. Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue. Eur. J. Biochem. 269: 5137–5148.[Medline]

Jackson, T.A., Xie, J., Yikilmaz, E., Miller, A.F., and Brunold, T.C. 2002. Spectroscopic and computational studies on iron and manganese superoxide dismutases: Nature of the chemical events associated with active-site pKs. J. Am. Chem. Soc. 124: 10833–10845.[CrossRef][Medline]

Jones, T.A., Zou, J.-Y., Cowan, S.W., and Kjeldgaard, M. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47: 110–115.

Kanematsu, S., and Asada, K. 1991. Chloroplast and cytosol isozymes of CuZn-superoxide dismutase: Their characteristic amino acid sequences. Free Radic. Res. Commun. 12–13: 383–390.

Kissinger, C.R., Gehlhaar, D.K., and Fogel, D.B. 1999. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D Biol. Crystallogr. 55: 484–491.[CrossRef][Medline]

Kliebenstein, D.J., Monde, R.A., and Last, R.L. 1998. Superoxide dismutase in Arabidopsis: An eclectic enzyme family with disparate regulation and protein localization. Plant Physiol. 118: 637–650.[Abstract/Free Full Text]

Kraulis, P. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24: 946–950.[CrossRef]

Lah, M.S., Dixon, M.M., Pattridge, K.A., Stallings, W.C., Fee, J.A., and Ludwig, M.L. 1995. Structure–function in Escherichia coli iron superoxide dismutase: Comparisons with the manganese enzyme from Thermus thermophilus. Biochemistry 34: 1646–1660.[CrossRef][Medline]

Maliekal, J., Karapetian, A., Vance, C., Yikilmaz, E., Wu, Q., Jackson, T., Brunold, T.C., Spiro, T.G., and Miller, A.F. 2002. Comparison and contrasts between the active site PKs of Mn-superoxide dismutase and those of Fe-superoxide dismutase. J. Am. Chem. Soc. 124: 15064–15075.[CrossRef][Medline]

Moews, P.C. and Kretsinger, R.H. 1975. Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis. J. Mol. Biol. 91: 201–228.[CrossRef][Medline]

Moran, J.F., James, E.K., Rubio, M.C., Sarath, G., Klucas, R.V., and Becana, M. 2003. Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules. Plant Physiol. 133: 1–10.[Free Full Text]

Muñoz, I.G., Moran, J.F., Becana, M., and Montoya, G. 2003. Crystallization and preliminary X-ray diffraction studies of the eukaryotic iron superoxide dismutase (FeSOD) from Vigna unguiculata. Acta Crystallogr. D Biol. Crystallogr. 59: 1070–1072.[CrossRef][Medline]

Murshudov, G.N., Vagin, A.A., Lebedev, A., Wilson, K.S., and Dodson, E.J. 1999. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D Biol. Crystallogr. 55: 247–255.[CrossRef][Medline]

Nicholls, A., Sharp, K.A., and Honig, B. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281–296.[CrossRef][Medline]

Ose, D.E. and Fridovich., I. 1976. Superoxide dismutase reversible removal of manganese and its substitution by cobalt, nickel or zinc. J. Biol. Chem. 251: 1217–1218.[Abstract/Free Full Text]

Otwinowski, Z. and Minor, W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307–326.

Ringe, D., Petsko, G.A., Yamakura, F., Suzuki, K., and Ohmori, D. 1983. Structure of iron superoxide dismutase from Pseudomonas ovalis at 2.9-Å resolution. Proc. Natl. Acad. Sci. 80: 3879–3883.[Abstract/Free Full Text]

Stallings, W.C., Powers, T.B., Pattridge, K.A., Fee, J.A., and Ludwig, M.L. 1983. Iron superoxide dismutase from Escherichia coli at 3.1-Å resolution: A structure unlike that of copper/zinc protein at both monomer and dimer levels. Proc. Natl. Acad. Sci. 80: 3884–3888.[Abstract/Free Full Text]

Stallings, W.C., Pattridge, K.A., Strong, R.K., and Ludwig, M.L. 1984. Manganase and iron superoxide dismutases are structural homologs. J. Biol. Chem. 259: 10695–10699.[Abstract/Free Full Text]

———. 1985. The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-Å resolution. J. Biol. Chem. 260: 16424–16432.[Abstract/Free Full Text]

Stallings, W.C., Metzger, A.L., Pattridge, K.A., Fee, J.A., and Ludwig, M.L. 1991. Structure–function relationships in iron and manganese superoxide dismutases. Free Radic. Res. Commun. 12–13: 259–268.

Tainer, J.A., Getzoff, E.D., Beem, K.M., Richardson, J.S., and Richardson, D.C. 1982. Determination and analysis of the 2 Å structure of copper, zinc superoxide dismutase. J. Mol. Biol. 160: 181–217.[CrossRef][Medline]

Vance, C.K. and Miller, A.F. 1998. Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase. Biochemistry 37: 5518–5527.[CrossRef][Medline]

Whittaker, M.M. and Whittaker, J.W. 1997. Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase. Biochemistry 36: 8923–8931.[CrossRef][Medline]

Yamakura, F. and Suzuki, K. 1980. Cadmium, chromium, and manganese replacement for iron in iron-superoxide dismutase from Pseudomonas ovalis. J. Biochem. 88: 191–196.[Abstract/Free Full Text]

Yikilmaz, E., Xie, J., Brunold, T.C., and Miller, A.F. 2002. Hydrogen-bond-mediated tuning of the redox potential of the non-heme Fe site of superoxide dismutase. J. Am. Chem. Soc. 124: 3482–3483.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muñoz, I. G.
Right arrow Articles by Montoya, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muñoz, I. G.
Right arrow Articles by Montoya, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS