Ferric nitrosylated myoglobin catalyzes peroxynitrite scavenging

Abstract

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ^·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ^·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ^·NOk_on) is (6.8 ± 0.7) × 10⁴ M⁻¹ s⁻¹ and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ^·NOk_off) is 3.1 ± 0.3 s⁻¹. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ^·NOk_off/^·NOk_on = (4.6 ± 0.7) × 10⁻⁵ M) is virtually the same as that directly measured (i.e., ^·NOK = (3.8 ± 0.5) × 10⁻⁵ M). In the absence of ^·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO₃⁻, the value of the second-order rate constant (i.e., ^Pk_on) being (1.9 ± 0.2) × 10⁴ M⁻¹ s⁻¹. However, in the presence of ^·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (^Pk_on* = (2.8 ± 0.3) × 10³ M⁻¹ s⁻¹). These data expand the multiple roles of ^·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ^·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.

Graphic abstract