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Iron(III)- and cobalt(III)-9,10,19,20-tetraphenylporphycenes, which possess bulky phenyl groups at the four meso positions of porphycene, were successfully incorporated into the haem acquisition protein HasA secreted by Pseudomonas aeruginosa. Crystal structure analysis revealed that loops surrounding the haem-binding site are highly flexible, remodelling themselves to accommodate bulky metal complexes with significantly different structures from the native haem cofactor.

The 22 kDa haem-binding protein, p22HBP, is highly expressed in erythropoietic tissues and binds to a range of metallo- and non-metalloporphyrin molecules with similar affinities, suggesting a role in haem regulation or synthesis. We have determined the three-dimensional solution structure of p22HBP and mapped the porphyrin-binding site, which comprises a number of loops and a alpha-helix all located on a single face of the molecule. The structure of p22HBP is related to the bacterial multi-drug resistance protein BmrR, and is the first protein with this fold to be identified in eukaryotes. Strikingly, the porphyrin-binding site in p22HBP is located in a similar position to the drug-binding site of BmrR. These similarities suggest that the broad ligand specificity observed for both BmrR and p22HBP may result from a conserved ligand interaction mechanism. Taken together, these data suggest that the both the fold and its associated function, that of binding to a broad range of small hydrophobic molecules, are ancient, and have been adapted throughout evolution for a variety of purposes.

We have designed and synthesized two series of amphiphilic two-α-helix peptides, that bound FeIII–mesoporphyrin (haem) through a ligation of two His residues. The interaction between the peptides and the haem was characterized by UV–VIS and circular dichroism (CD) measurements. The first series of peptides, designed on the basis of the coiled-coil motif, showed a unique haem binding property which was dependent on the concentration of trifluoroethanol (TFE) present. The peptides bound the haem effectively only when the two-α-helix structures were controlled by the addition of 10–25% TFE. These results indicated that the haem binding ability of the peptides could be regulated by the change in peptide conformation with TFE. The second series of two-α-helix peptides, designed on the basis of the amphiphilic α-helix motif, but not of the coiled-coil motif, formed an α-helix structure and bound the haem in a buffer. Furthermore, in the presence of peptides, the haem showed strong induced CD peaks at the Soret region, implying that the haem chromophore was highly oriented in the peptide structures. The catalytic activity of the haem bound to the peptides, which was similar to that of peroxidase, was significantly depressed with increased binding constants and the Soret-CD intensities. It was demonstrated that the catalytic activity of the haem was correlated with the rigidity and orientation of the b-type haem in the polypeptides.

9 páginas, 7 figuras, 2 tablas -- PAGS nros. 690-698, The reactive oxygen species hydrogen peroxide is a byproduct of the -oxidation process that occurs in peroxisomes. Since reactive oxygen species can cause serious damage to biomolecules, a number of scavengers control their intracellular levels. One such scavenger that is present in the peroxisome is the oxidoreductase catalase. In this study, the crystal structure of heterologously expressed peroxisomal catalase from the thermotolerant yeast Hansenula polymorpha has been determined at 2.9 Å resolution. H. polymorpha catalase is a typical peroxisomal catalase; it is tetrameric and is highly similar to catalases from other organisms. However, its hydrogen peroxide-degrading activity is higher than those of a number of other catalases for which structural data are available. Structural superimpositions indicate that the nature of the major channel, the path for hydrogen peroxide to the active site, varies from those seen in other catalase structures, an observation that may account for the high activity of H. polymorpha catalase, This project was partially supported by a Rubicon Fellowship from the Netherlands Organization for Scientific Research (NWO) awarded to CW (825.08.023) and performed under the PhD program `Structure and Function of Proteins' from the Universitat Autònoma de Barcelona (UAB)

Entamoeba histolytica is a human pathogen which can grow using different sources of iron such as free iron, lactoferrin, transferrin, ferritin or haemoglobin. In the present study, we found that E. histolytica was also capable of supporting its growth in the presence of haem as the sole iron supply. In addition, when trophozoites were maintained in cultures supplemented with haemoglobin as the only iron source, the haem was released and thus it was introduced into cells. Interestingly, the Ehhmbp26 and Ehhmbp45 proteins could be related to the mechanism of iron acquisition in this protozoan, since they were secreted to the medium under iron-starvation conditions, and presented higher binding affinity for haem than for haemoglobin. In addition, both proteins were unable to bind free iron or transferrin in the presence of haem. Taken together, our results suggest that Ehhmbp26 and Ehhmbp45 could function as haemophores, secreted by this parasite to facilitate the scavenging of haem from the host environment during the infective process.

The authors acknowledge financial support from Fundacao para a Ciencia e Tecnologia, Portugal through grant SFRH/BPD/30239/2006 and project PTDC/QUI/64203/2006. The authors also acknowledge the ESRF, Grenoble (beamlines ID14- EH2, ID14- EH4, ID23- EH1 and ID-29) for access and technical support during data collection. Human SOUL (hSOUL) is a 23 kDa haem-binding protein that was first identified as the PP23 protein isolated from human full-term placentas. Here, the overexpression, purification and crystallization of hSOUL are reported. The crystals belonged to space group P6422, with unit-cell parameters a = b = 145, c = 60 Å and one protein molecule in the asymmetric unit. X-ray diffraction data were collected to 3.5 Å resolution at the ESRF. A preliminary model of the three-dimensional structure of hSOUL was obtained by molecular replacement using the structures of murine p22HBP (PDB codes 2gov and 2hva), obtained by solution NMR, as search models. publishersversion published

Based on a simplified model of the all-alpha class of protein, all packing arrangements of alpha-helices were generated and assessed by both general and specific structural rules. The method was applied to myoglobin and parvalbumin, which were both ranked in the top 4% of folds under the general packing constraints. Incorporation of the restrictions implied by the EF-hand motifs of parvalbumin were sufficient to select the correct fold as one of two (equal scoring) possibilities. Myoglobin scored well under the general packing constraints and the addition of a single distance constraint, implied by haem binding, was sufficient to select the correct fold as one of several candidates. Incorporation of a score for complementary hydrophobic packing between helices further selected myoglobin as a unique fold but did not improve the ranking of parvalbumin. For both proteins, the alpha-helices were predicted from multiply aligned sequences using pattern-matching methods and no specific aspect of the known X-ray structures influenced this or the prediction of the correct folds. Although the method is currently of limited generality, its further applications and extension to a more detailed structural level are discussed.

The thermodynamics of ferric and ferrous haem affinity of a de novo designed four-alpha-helix bundle protein and the associated haem electrochemistry is described.

A nucleic acid binding protein, rop, has conserved topology with a number of redox proteins; this is exploited to engineer haem binding, expanding its function as a redox protein.

Rhodnius haem-binding protein (RHBP) from the bloodsucking insect Rhodnius prolixus, a 15 kDa protein, has been crystallized using polyethylene glycol as a precipitant. X-ray diffraction data have been collected at a synchrotron source. The crystals belong to the space group P4(1(3))2(1)2, with unit-cell parameters a = b = 64.98, c = 210.68 A, and diffract beyond 2.6 A resolution.

Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems. We have solved this structure in a tetragonal crystal form at 2.9 A resolution. We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits. The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits. The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits. A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment. This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.

A designed peptide H2α17-I bound FeIII–mesoporphyrin (haem); and the haem-binding prevented the peptide forming β-sheet aggregates by facilitating the formation of an α-helix tetramer, indicating that the folding state of artificially designed peptides could be regulated by cofactor binding.

A designed two-α-helix peptide H2α-17 bound effectively FeIII–mesoporphyrin (haem), and the haem binding simultaneously induced the molecular assembly of the peptide from a monomeric to a tetrameric form.

A designed two-α-helix peptide His-2α binds effectively Fe III –mesoporphyrin (haem) and enhances N-demethylase activity of the haem, when the 2α-helix structure is annealed by the addition of 10–20% trifluoroethanol.

Horse spleen ferritin, a spherical protein shell of 24 subunits, contains no haem when extracted. This contrasts with ferritins isolated from bacterial sources which have the capacity to bind up to 24 haem groups [(1990) FEBS Lett. 271, 141–143] via two methionine residues [(1990) Nature 341, 771]. Here it is shown that horse spleen ferritin can bind between 15 and 17 haems per 24 subunits with an apparent association constant of 2.2–3.2 × 104 M−1. The strength of haem binding appears to be unaffected either by the presence of the core or by the oxidation state of the haem. The demonstration of the ability of animal ferritin to bind haem strengthens the similarity between it and bacterioferritin and could have major consequences for its mechanism of action in physiological iron uptake and release processes.

These two phenomena are involved in the instability of haemoglobins associated with hereditary Heinz body anaemia.