Time filter

Source Type

Ascenzi P.,Third University of Rome | Coletta M.,University of Rome Tor Vergata | Coletta M.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | Wilson M.T.,University of Essex | And 5 more authors.
IUBMB Life | Year: 2015

Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc. © 2015 IUBMB Life, 67(2):98-109, 2015 © 2015 International Union of Biochemistry and Molecular Biology.


Ascenzi P.,Third University of Rome | Ascenzi P.,National Institute for Infectious Diseases | Bolli A.,Third University of Rome | Di Masi A.,Third University of Rome | And 6 more authors.
Journal of Biological Inorganic Chemistry | Year: 2011

Human serum heme-albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA-heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K 0 = (3.9 ± 0.4) × 10-4 and (1.3 ± 0.1) × 10-5 M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K h = (4.3 ± 0.4) × 10-3 and (1.2 ± 0.1) × 10-4 M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H 0 = (1.9 ± 0.2) × 10-8 M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H d = (2.1 ± 0.2) × 10 -7 M]. In the absence and presence of CO2, the values of the second-order rate constant (l on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 105 and 4.3 × 10 5 M-1 s-1, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO2. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow's site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity. © 2010 SBIC.


Ascenzi P.,Third University of Rome | Bolli A.,Third University of Rome | Gullotta F.,University of Rome Tor Vergata | Gullotta F.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | And 2 more authors.
IUBMB Life | Year: 2010

Heme endows human serum albumin (HSA) with globin-like reactivity and spectroscopic properties. Here, the effect of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitrite isomerization to NO3- by ferric HSA-heme (HSA-heme-Fe(III)) is reported. Drugs binding to Sudlow's site I impair dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III). The allosteric modulation of HSA-heme-Fe(III)-mediated peroxynitrite isomerization by drugs has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I or protrudes into the heme cleft (i.e., the fatty acid site 1, FA1), depending on ligand occupancy of either sites. © 2010 IUBMB.


Ascenzi P.,Third University of Rome | Ascenzi P.,National Institute For Infectious Diseases Irccs Lazzaro Spallanzani | Cao Y.,Third University of Rome | Cao Y.,University of Rome Tor Vergata | And 7 more authors.
FEBS Journal | Year: 2010

Heme endows human serum albumin (HSA) with heme-protein-like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme-albumin [HSA-heme-Fe(III)] are reported. All data were obtained at 20 °C. At pH 5.5, HSA-heme-Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA-heme-Fe(III) [HSA-heme-Fe(III)-NO]. By contrast, at pH ≥ 6.5, the addition of NO to HSA-heme-Fe(III) leads to the transient formation of HSA-heme-Fe(III)-NO in equilibrium with HSA-heme-Fe(II)-NO+. Then, HSA-heme-Fe(II)-NO+ undergoes nucleophilic attack by OH- to yield ferrous human serum heme-albumin [HSA-heme-Fe(II)]. HSA-heme-Fe(II) further reacts with NO to give nitrosylated HSA-heme-Fe(II) [HSA-heme-Fe(II)-NO] . The rate-limiting step for reductive nitrosylation of HSA-heme-Fe(III) is represented by the OH--mediated reduction of HSA-heme-Fe(II)-NO + to HSA-heme-Fe(II). The value of the second-order rate constant for OH--mediated reduction of HSA-heme-Fe(II)-NO+ to HSA-heme-Fe(II) is 4.4 × 103 m-1·s -1. The present results highlight the role of HSA-heme-Fe in scavenging reactive nitrogen species. © 2010 FEBS.


Di Masi A.,Third University of Rome | Gullotta F.,University of Rome Tor Vergata | Gullotta F.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | Bolli A.,Third University of Rome | And 3 more authors.
FEBS Journal | Year: 2011

The ibuprofen primary binding site FA3-FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1-Glu382 truncated HSA (tHSA)-heme-Fe(III) and nitrosylated tHSA-heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA-heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA-heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA-heme and HSA-heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3-FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA. © 2011 The Authors Journal compilation © 2011 FEBS.


Ascenzi P.,Third University of Rome | Ascenzi P.,CNR Institute of Neuroscience | Tundo G.R.,University of Rome Tor Vergata | Tundo G.R.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | And 4 more authors.
Journal of Biological Inorganic Chemistry | Year: 2013

Human serum heme-albumin (HSA-heme-Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA-heme-Fe [HSA-heme-Fe(II)] is reported. The value of the second-order rate constant for the reduction of NO- 2 to NO and the concomitant formation of nitrosylated HSA-heme-Fe(II) (i.e., kon) is 1.3 M-1 s-1 at pH 7.4 and 20 °C. Values of kon increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA-heme-Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA-heme-Fe(II) is (3.1 ± 0.4) × 10-4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the NO- 2 reductase activity of HSA-heme-Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(-heme-Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity. © SBIC 2013.


Di Masi A.,Third University of Rome | Leboffe L.,Third University of Rome | Trezza V.,Third University of Rome | Fanali G.,University of Insubria | And 4 more authors.
Current Pharmaceutical Design | Year: 2015

Human serum albumin (HSA) represents an important determinant of plasma oncotic pressure and a relevant factor that modulates fluid distribution between the body compartments. Moreover, HSA (i) represents the depot and transporter of several compounds, both endogenous and exogenous, (ii) affects the pharmacokinetics of many drugs, (iii) regulates chemical modifications of some ligands, (iv) shows (pseudo-)enzymatic properties, (v) inactivates some toxic compounds, and (vi) displays anti-oxidant properties. HSA binding and (pseudo-)enzymatic properties are regulated competitively, allosterically, and by covalent modifications. While competitive inhibition of HSA binding properties is evident, allosteric mechanisms and covalent modifications affecting HSA reactivity are less clear. In several pathological conditions in which free heme-Fe levels increase, the buffering capacity of plasma hemopexin is overwhelmed and most of heme-Fe binds to the fatty acid site 1 of HSA. HSA-heme-Fe displays globin-like properties; in turn, heme-Fe modulates competitively and allosterically HSA binding and reactivity properties. Remarkably, heme-Fe-mediated HSA properties are time-dependent, representing a case for “chronosteric effects”. Here, we review the drug-based modulation of (i) heme-Fe-recognition by HSA and (ii) heme-Fe-mediated reactivity. © 2015 Bentham Science Publishers.


Ascenzi P.,Third University of Rome | Di Masi A.,Third University of Rome | Tundo G.R.,University of Rome Tor Vergata | Tundo G.R.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | And 4 more authors.
PLoS ONE | Year: 2014

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O 2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (CjtrHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous MttrHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo. © 2014 Ascenzi et al.


Tundo G.,University of Rome Tor Vergata | Ciaccio C.,University of Rome Tor Vergata | Sbardella D.,University of Rome Tor Vergata | Sbardella D.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | And 5 more authors.
PLoS ONE | Year: 2012

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. © 2012 Tundo et al.


Ascenzi P.,Third University of Rome | Marino M.,Third University of Rome | Ciaccio C.,University of Rome Tor Vergata | Ciaccio C.,Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems | And 3 more authors.
IUBMB Life | Year: 2014

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 (h OH-=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.

Loading Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems collaborators
Loading Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems collaborators