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., ·NOkon) 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., ·NOkoff) is 3.1 ± 0.3 s-1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (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., Pkon) 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 (Pkon* = (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.