Chiara Ciaccio

University of Rome Tor Vergata, Roma, Latium, Italy

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Publications (45)160.16 Total impact

  • PLoS ONE 06/2015; 10(6):e0125959. DOI:10.1371/journal.pone.0125959 · 3.23 Impact Factor
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    ABSTRACT: The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2- binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2- concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2-binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.
    PLoS ONE 05/2015; 10(5):e0125005. DOI:10.1371/journal.pone.0125005 · 3.23 Impact Factor
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    ABSTRACT: The properties of three novel Platinum(II) compounds toward the insulin-degrading enzyme (IDE) enzymatic activity have been investigated under physiological conditions. The rationale of this study resides on previous observations that these compounds, specifically designed and synthesized by some of us, induce apoptosis in various cancer cell lines, whereas IDE has been proposed as a putative oncogene involved in neuroblastoma onset and progression. Two of these compounds, namely [PtCl(O,O′-acac)(DMSO)] and [Pt(O,O′-acac)(γ-acac)(DMS)], display a modulatory behavior, wherefore activation or inhibition of IDE activity occurs over different concentration ranges (suggesting the existence of two binding sites on the enzyme). On the other hand, [Pt(O,O′-acac)(γ-acac)(DMSO)] shows a typical competitive inhibitory pattern, characterized by a meaningful affinity constant (K i = 0.95 ± 0.21 μM). Although all three compounds induce cell death in neuroblastoma SHSY5Y cells at concentrations exceeding 2 μM, the two modulators facilitate cells’ proliferation at concentrations ≤ 1.5 μM, whereas the competitive inhibitor [Pt(O,O′-acac)(γ-acac)(DMSO)] only shows a pro-apoptotic activity at all investigated concentrations. These features render the [Pt(O,O′-acac)(γ-acac)(DMSO)] a promising “lead compound” for the synthesis of IDE-specific inhibitors (not characterized yet) with therapeutic potentiality.
    JBIC Journal of Biological Inorganic Chemistry 12/2014; 20(1). DOI:10.1007/s00775-014-1217-3 · 3.16 Impact Factor
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    ABSTRACT: Native horse heart cytochrome c (cytc) displays a very low reactivity toward ligands and does not exhibit catalytic properties. However, upon bovine cardiolipin (CL) binding, cytc achieves myoglobin-like properties. Here, NO binding to CL-cytc(III) between pH 7.2 and 9.5, at 20 °C, is reported. At pH 7.2, CL-cytc(III) undergoes reversible nitrosylation, whereas between pH 7.9 and 9.5 CL-cytc(III) undergoes irreversible reductive nitrosylation leading to the formation of CL-cytc(II)-NO. Over the whole pH range explored, first-order kinetics of NO binding to CL-cytc(III) (k = 9.3 s−1) indicates that ligand binding is limited by the cleavage of the weak heme-Fe distal bond. Between pH 7.9 and 9.5, nitrosylated CL-cytc(III) converts to the ligand-free ferrous derivative (CL-cytc(II)), this process being pH-dependent (hOH− = 3.0 × 103 M−1 s−1). Then, CL-cytc(II) converts to nitrosylated CL-cytc(II), in the presence of NO excess. The value of the second-order rate constant for CL-cytc(II) nitrosylation is essentially pH-independent, the average value of lon being 1.4 × 107 M−1 s−1. These results agree with the view that CL-cytc nitrosylation may play a role in apoptosis regulation. © 2014 IUBMB Life, 00(00):000–000, 2014
    International Union of Biochemistry and Molecular Biology Life 06/2014; 66. DOI:10.1002/iub.1283 · 2.76 Impact Factor
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    ABSTRACT: Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO2- to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are kapp1 = 9.6±0.2 M-1 s-1 and kapp2 = 1.2±0.1 M-1 s-1 (at pH 7.4 and 20°C). The kapp1 and kapp2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are happ = 3.8×104 M-1 s-1 and h0 = 2.8×10-1 s-1 (at pH 7.4 and 20°C). The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.
    PLoS ONE 05/2014; 9(5):e95391. DOI:10.1371/journal.pone.0095391 · 3.23 Impact Factor
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    ABSTRACT: Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10(-4) M, k on = (5.3±0.6)×10(3) M(-1) s(-1) and k off = (4.6±0.5)×10(-1) s(-1); and D = (1.2±0.2)×10(-3) M, d on = (1.3±0.4)×10(3) M(-1) s(-1), and d off = 1.5±0.4 s(-1), respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.
    PLoS ONE 08/2013; 8(8):e69762. DOI:10.1371/journal.pone.0069762 · 3.23 Impact Factor
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    ABSTRACT: Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O2, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.
    PLoS ONE 06/2013; 8(6):e66144. DOI:10.1371/journal.pone.0066144 · 3.23 Impact Factor
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    ABSTRACT: Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II).
    PLoS ONE 03/2013; 8(3):e58842. DOI:10.1371/journal.pone.0058842 · 3.23 Impact Factor
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    ABSTRACT: Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(E20)101 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
    Biochimica et Biophysica Acta 02/2013; 1834(9). DOI:10.1016/j.bbapap.2013.02.026 · 4.66 Impact Factor
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    ABSTRACT: Methanosarcina acetivorans is a strictly anaerobic non-motile methane-producing Archaea expressing protoglobin (Pgb) which might either facilitate O(2) detoxification or act as a CO sensor/supplier in methanogenesis. Unusually, Methanosarcina acetivorans Pgb (MaPgb) binds preferentially O(2) rather than CO and displays anticooperativity in ligand binding. Here, kinetics and/or thermodynamics of ferric and ferrous MaPgb (MaPgb(III) and MaPgb(II), respectively) nitrosylation are reported. Data were obtained between pH 7.2 and 9.5, at 22.0 °C. Addition of NO to MaPgb(III) leads to the transient formation of MaPgb(III)-NO in equilibrium with MaPgb(II)-NO(+). In turn, MaPgb(II)-NO(+) is converted to MaPgb(II) by OH(-)-based catalysis. Then, MaPgb(II) binds NO very rapidly leading to MaPgb(II)-NO. The rate-limiting step for reductive nitrosylation of MaPgb(III) is represented by the OH(-)-mediated reduction of MaPgb(II)-NO(+) to MaPgb(II). Present results highlight the potential role of MaPgb in scavenging of reactive nitrogen and oxygen species.
    Biochemical and Biophysical Research Communications 12/2012; 430(4). DOI:10.1016/j.bbrc.2012.11.122 · 2.28 Impact Factor
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    ABSTRACT: Insulin-Degrading Enzyme (IDE) is a highly conserved zinc metallopeptidase which is ubiquitously distributed in human tissues, being particularly abundant in the brain, liver and muscles. IDE activity has been historically associated to insulin and β-amyloid catabolism. However, over the last decade, several experimental findings have established that IDE is also involved in a wide variety of physiopathological processes, including ubiquitin clearance and Varicella Zoster Virus infection. In this study, we demonstrate that normal and malignant cells exposed to different stresses markedly up-regulate IDE in a Heat Shock Protein (HSP)-like fashion. Additionally, we focused our attention on tumor cells and report that (i) IDE is overexpressed in vivo in tumors of the Central Nervous System (CNS); (ii) IDE-silencing inhibits neuroblastoma (SHSY5Y) cell prolifera- tion and triggers cell death; (iii) IDE inhibition is accompanied by a decrease of the poly-ubiquitinated protein content and co-immunoprecipitates with proteasome and ubiquitin in SHSY5Y cells. In this work, we propose a novel role for IDE as a Heat-Shock Protein with implications in cell growth regulation and cancer progression, thus opening up an intriguing hypothesis of IDE as an anticancer target.
    Journal of Biological Chemistry 11/2012; 288(4). DOI:10.1074/jbc.M112.393108 · 4.57 Impact Factor
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    Biochemical and Biophysical Research Communications 07/2012; 424(1):202. DOI:10.1016/j.bbrc.2012.06.001 · 2.28 Impact Factor
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    ABSTRACT: Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimer's disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752-2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20-30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II).
    Journal of inorganic biochemistry 06/2012; 117. DOI:10.1016/j.jinorgbio.2012.06.010 · 3.27 Impact Factor
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    ABSTRACT: The deposition of β-amyloid (Aβ) into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD). Insulin-degrading-enzyme (IDE) is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.
    PLoS ONE 04/2012; 7(4):e34376. DOI:10.1371/journal.pone.0034376 · 3.23 Impact Factor
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    ABSTRACT: Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin with peculiar structural properties such as a completely buried haem and two orthogonal tunnels connecting the distal cavity to the solvent. CO binding to and dissociation from MaPgb occur through a biphasic kinetics. We show that the heterogenous kinetics arises from binding to (and dissociation from) two tertiary conformations in ligation-dependent equilibrium. Ligation favours the species with high binding rate (and low dissociation rate). The equilibrium is shifted towards the species with low binding (and high dissociation) rates for the unliganded molecules. A quantitative model is proposed to describe the observed carbonylation kinetics.
    PLoS ONE 03/2012; 7(3):e33614. DOI:10.1371/journal.pone.0033614 · 3.23 Impact Factor
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    ABSTRACT: Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.
    Molecular Aspects of Medicine 11/2011; 33(2):119-208. DOI:10.1016/j.mam.2011.10.015 · 10.30 Impact Factor
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    ABSTRACT: Carboxymethylation of equine heart cytochrome c (cytc) changes its tertiary structure by disrupting the heme-Fe-Met80 distal bond, such that carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) on peroxynitrite isomerization by ferric CM-cytc (CM-cytc-Fe(III)) is reported. Unlike native ferric cytc (cytc-Fe(III)), CM-cytc-Fe(III) catalyzes peroxynitrite isomerization, the value of the second order rate constant (k(on)) is 6.8 × 10(4)M(-1)s(-1). However, CM-cytc-Fe(III) is less effective in peroxynitrite isomerization than CL-bound cytc-Fe(III) (CL-cytc-Fe(III); k(on)=3.2 × 10(5)M(-1)s(-1)). Moreover, CL binding to CM-cytc-Fe(III) facilitates peroxynitrite isomerization (k(on)=5.3 × 10(5)M(-1)s(-1)). Furthermore, the value of the dissociation equilibrium constant for CL binding to CM-cytc-Fe(III) (K=1.8 × 10(-5)M) is lower than that reported for CL-cytc-Fe(III) complex formation (K=5.1 × 10(-5)M). Although CM-cytc-Fe(III) and CL-cytc-Fe(III) display a different heme distal geometry and heme-Fe(III) reactivity, the heme pocket and the CL cleft are allosterically linked.
    Biochemical and Biophysical Research Communications 10/2011; 415(3):463-7. DOI:10.1016/j.bbrc.2011.10.094 · 2.28 Impact Factor
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    ABSTRACT: Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.
    European Journal of Biochemistry 09/2011; 17(1):133-47. DOI:10.1007/s00775-011-0837-0 · 3.16 Impact Factor
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    Biochemical and Biophysical Research Communications 08/2011; 412(1):194. DOI:10.1016/j.bbrc.2011.07.095 · 2.28 Impact Factor
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    ABSTRACT: The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed.
    International Union of Biochemistry and Molecular Biology Life 07/2011; 63(7):566-73. DOI:10.1002/iub.492 · 2.76 Impact Factor