title: Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme-albumin.
abstract: 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 CO(2), the values of the second-order rate constant (l (on)) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10(5) 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 CO(2). 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.
authors.affiliation: Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, Rome, Italy. ascenzi@uniroma3.it
authors.forename: Paolo
authors.initials: P
authors.lastname: Ascenzi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bolli
authors.forename: Alessandra
authors.initials: A
authors.lastname: di Masi
authors.forename: Grazia R
authors.initials: GR
authors.lastname: Tundo
authors.forename: Gabriella
authors.initials: G
authors.lastname: Fanali
authors.forename: Massimo
authors.initials: M
authors.lastname: Coletta
authors.forename: Mauro
authors.initials: M
authors.lastname: Fasano
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D006418:Heme / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D007538:Isoniazid / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D030421:Peroxynitrous Acid / Q000737:chemistry*; D012293:Rifampin / Q000737:chemistry / Q000494:pharmacology*; D012709:Serum Albumin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D012709:Serum Albumin; D030421:Peroxynitrous Acid; D006418:Heme; D007538:Isoniazid; D012293:Rifampin