Maddalena Cross

Metal-substituted derivatives of the rubredoxin from Clostridium pasteurianum.

Five different metal-substituted forms of Clostridium pasteurianum rubredoxin have been prepared and crystallized. The single Fe atom present in the Fe(S-Cys)(4) site of the native form of the protein was exchanged in turn for Co, Ni, Ga, Cd and Hg. All five forms of rubredoxin crystallized in space group R3 and were isomorphous with the native protein. The Co-, Ni- and Ga-substituted proteins exhibited metal sites with geometries similar to that of the Fe form (effective D(2d) local symmetry), as did the Cd and Hg proteins, but with a significant expansion of the metal-sulfur bond lengths. A knowledge of these structures contributes to a molecular understanding of the function of this simple iron-sulfur electron-transport protein.

Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum : solvent access versus structural changes as determinants of reversible potential

Abstract The Pri sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pri), L (Bui) and I (Bus) at those positions. X-ray crystal structures of the FeIII forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the sidechains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH···Sγ hydrogen bond at the active site and lead to a shift in potentialof +50 mV.

Women's satisfaction with antenatal care: comparing women in Sweden and Australia.

BACKGROUND Satisfaction with antenatal care could differ depending on the organisation and the context of care. AIM To compare antenatal care in Australia and Sweden, to identify deficiencies in the content of antenatal care and what aspects contributed most in dissatisfaction with antenatal care. METHODS A longitudinal survey of 123 Australian and 386 Swedish women recruited during one year in regional hospitals in Sweden and Australia. Data collected by three questionnaires. RESULTS Women in Australia had more antenatal visits, less continuity of midwife caregiver but were more satisfied with antenatal education and the emotional aspects of antenatal care. Although the overall satisfaction was high, deficiencies were found in more than half of the studied variables in the content of care. Women in Sweden were more dissatisfied with information about labour and birth (OR 3.1; 1.8-5.3) and information about the time following birth (OR 3.8; 2.2-6.3), but more satisfied with the involvement of the father (OR 0.3; 0.2-0.6). Factors that contributed most to dissatisfaction with antenatal care overall were deficiency in information about pregnancy related issues (OR 3.4; 1.3-8.7) and not being taken seriously by the midwife (OR 4.1; 1.6-10.1). CONCLUSION Satisfaction with antenatal care was high in both groups of women. Australian women were more satisfied than the Swedish women with the emotional aspects of care. Deficiencies were found in more than half of the variables measured relating to the specific aspects of care. Lack of information and not being treated seriously were important factors for not being satisfied.

Psychometric evaluation and refinement of the Prenatal Attachment Inventory

Objective: To explore the underlying structure of the Prenatal Attachment Inventory (PAI) and to assess the psychometric properties of the subscales. Background: The establishment of the mother–fetus relationship is an important developmental task for successful adaption to pregnancy, with implications for the well-being of mother and child. The PAI was developed to measure the relationship between a women and her fetus. Although originally developed as a single dimension, other researchers have suggested alternative factor structures. Methods: A self-report questionnaire, including the PAI, was administered to 775 Swedish women in late pregnancy. Psychometric assessment of the PAI was undertaken using exploratory (EFA) and confirmatory factor analysis (CFA), and Rasch analysis. Results: EFA indicated a three-factor solution, which was confirmed by CFA. The previously reported single-factor and five-factor solutions were not supported. Rasch analysis was used to form three 6-item PAI-Revised subscales (Anticipation, Interaction, Differentiation). All subscales showed good overall fit to the Rasch model and good internal consistency. The three subscales were moderately intercorrelated, sharing between 23% and 42% of their variance, suggesting they should be used separately, pending further research concerning their unique predictive power. Conclusion: These results suggest that PAI-Revised is a psychometrically sound tool, suitable for use in research and clinical settings. It can be used to guide the identification, support, and follow-up of pregnant women with low attachment. The three subscales, although related, may tap different aspects of the attachment construct, with different antecedents and consequences for the well-being of mother and child.

Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe(III)(S-Cys)3(OH) centre in the C6S mutant.

AbstractRedox thermodynamic data provide a detailed insight into control of the reduction potential E°′ of the [Fe(S-Cys)4] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of ~200 mV in E°′ observed for the [Fe(S-Cys)3(O-Ser)]–/2– couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate. However, the pH dependence of the potentials below characteristic pKared≈7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer FeII-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKared≥9.3, but for pH<pKared the entropic term again becomes significant. The available data for the internal ligand mutant C39S appear to follow those for the surface ligand mutants. A longer FeIII-O-Ser link in the oxidized form is expected from structural considerations and a smaller decrease of ~100 mV in E°′ is observed for this particular [Fe(S-Cys)3(O-Ser)]–/2– couple. The reduced form is particularly susceptible to hydrolysis with consequent irreversible electrochemistry, an apparent consequence of an even longer FeII-O(H)-Ser bond. The second internal ligand mutant C6S exhibits starkly different behaviour. The cause was traced to the presence of a FeIII(S-Cys)3(OH)– centre in the oxidized form. Resonance Raman spectroscopy on 54Fe-, H218O- and D2O-enriched samples supports the presence of an iron hydroxyl group that donates a hydrogen bond to the OH group of the free S6 side chain and/or a cluster of bound water molecules. The thermodynamic parameters can then be rationalized in terms of the following processes: pH>pKa∼9: [FeIII(S-Cys)3(OH)]– + e– → [FeII(S-Cys)3(OH)]2– pH<pKa: [FeIII(S-Cys)3(OH)]– + H+ + e– → [FeII(S-Cys)3(OH2)]–

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)3(OH) centres

Abstract. The electron transfer protein rubredoxin from Clostridiumpasteurianum contains an Fe(S-Cys)4 active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichiacoli. The C42A protein expresses with a FeIII2S2 cluster in place. In contrast, the other proteins are isolated in colourless forms, although a FeIII2S2 cluster may be assembled in the C42G protein via incubation with FeIII and sulfide. The four mutant proteins were isolated as stable mononuclear HgII forms which were converted to unstable mononuclear FeIII preparations that contain both holo and apo protein. The FeIII systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant FeIII form in the C9A preparation is a Fe(S-Cys)3(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)3(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate FeIII form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0355-1.

Application of NMRD to Hydration of Rubredoxin and a Variant Containing a (Cys-S)3FeIII(OH) Site

Hydration of oxidized rubredoxin (Fe(III)(S-Cys)(4) center) was investigated by (1)H and (17)O relaxation measurements of bulk water as a function of the applied magnetic field (nuclear magnetic relaxation dispersion). Oxidized rubredoxin showed an increased water (1)H relaxation profile with respect to the diamagnetic gallium derivative or reduced species. Analysis of the data shows evidence of exchangeable proton(s) approximately 4.0-4.5 A from the metal ion, the exchange time being longer than 10(-10) s and shorter than 10(-5) s. The correlation time for the proton-electrons interaction is 7 x 10(-11) s and is attributed to the effective electron relaxation time. Its magnitude is consistent with the large signal linewidths of the protein donor nuclei, observed in high resolution NMR spectra. For reduced rubredoxin, such correlation time is proposed to be smaller than 10(-11) s. (17)O relaxation measurements suggest the presence of at least one long-lived protein-bound water molecule. Analogous relaxation measurements were performed on the C6S rubredoxin variant, whose iron(III) center has been previously shown to be coordinated to three cysteine residues and a hydroxide ion above pH 6. (1)H nuclear magnetic relaxation dispersion profiles indicate increased hydration with respect to the wild-type.

Metal-substituted derivatives of the rubredoxin from Clostridium pasteurianum. Addendum

Five different metal-substituted forms of Clostridium pasteurianum rubredoxin have been prepared and crystallized. The single Fe atom present in the Fe(S-Cys)(4) site of the native form of the protein was exchanged in turn for Co, Ni, Ga, Cd and Hg. All five forms of rubredoxin crystallized in space group R3 and were isomorphous with the native protein. The Co-, Ni- and Ga-substituted proteins exhibited metal sites with geometries similar to that of the Fe form (effective D(2d) local symmetry), as did the Cd and Hg proteins, but with a significant expansion of the metal-sulfur bond lengths. A knowledge of these structures contributes to a molecular understanding of the function of this simple iron-sulfur electron-transport protein.