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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.


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.


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.


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.

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