Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217

REASSESSMENT OF THE MÖSSBAUER SPECTRA OF IRON(I I) PYRIDINE-N-OXIDE PERCHLORATE : EVIDENCE FOR SITE DISTORTIONS ENHANCED BY GRINDING

Les spectres Mössbauer de poudre de Fe(C5H5NO)6 (ClO4)2 mettent en évidence l'existence de relaxations électroniques lentes à basse température et en champ nul, mais aucune structure magnétique hyperfine n'a pu être observée. Ces spectres sont bien compris à partir d'un modèle où l'effet dominant est dû aux conséquences du traitement mécanique de mise en poudre sur les sites de Fe2+ qui sont des octaèdres en distorsion trigonale. Le traitement mécanique introduit une distorsion nonaxiale supplémentaire qui est représentée par un terme B22O22 dans l'Hamiltonien du champ cristallin. Une distribution des valeurs du paramètre B22 permet une meilleure simulation, avec un pic à B22 ≈ 0.3 cm-1 alors que pour le matériau massif le pic de distribution se trouve à B22 = 0.57Fe Mössbauer spectra of crushed powders of Fe(C5H5NO)6(ClO4)2 show evidence of slow electronic relaxation at low temperature in zero applied magnetic field, but resolved magnetic hyperfine structure is not observed. However, the spectra of small uncrushed crystals contain well-resolved magnetic hyperfine structure. These spectra have been well reproduced using a mode1 in which the dominant effect of the mechanical crushing on the Fe2+ site, which is a trigonally distorted octahedron, is to introduce a non-axial distortion that is represented in the crystal field Hamiltanian by a term B22O22.Distributions of values of the parameter B22 enabled better simulation of the spectra in both cases, but while the distribution required for the spectrum of the uncrushed crystals had a strong peak at B22 = O, that for the powder was peaked at B22 ~ 0.3 cm-1

Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c

His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c. Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability. The His26Tyr mutant shows properties typical of the molten globule. Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein. The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein. Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine. Furthermore, a reduction in catalytic activity indicates that the functional properties are altered. The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44. Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein. In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH. Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect. This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80. By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant. This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c

High protein structural flexibility of a truncated hemoglobin from an antarctic cold-adapted bacterium

Fil:Boechi, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Estrin, D.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina