Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme-albumin

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 degrees C. The affinity of isoniazid and rifampicin for HSA [K-0 = (3.9 +/- 0.4) x 10(-4) and (1.3 +/- 0.1) x 10(-5) M, respectively] decreases by about I order of magnitude upon heme-Fe(III) binding to HSA [K-h = (4.3 +/- 0.4) x 10(-3) and (1.2 +/- 0.1) x 10(-4) M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H-0 = (1.9 +/- 0.2) x 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) x 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 x 10(5) and 4.3 x 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! 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.