Allosteric modulation by S-nitrosation in the low-O2 affinity myoglobin from rainbow trout

Myoglobin (Mb) serves in the facilitated diffusion and storage of O2 in heart and skeletal muscle, where it also regulates O2 consumption via nitric oxide (NO) scavenging or generation. S-nitrosation at reactive cysteines may generate S-nitroso Mb (Mb-SNO) and contribute further to NO homeostasis. In being a monomer, Mb is commonly believed to lack allosteric control of heme reactivity. Here, we test whether in rainbow trout, a fast swimmer living in well-aerated water, the Mb-O2 affinity is regulated by ionic cofactors and S-nitrosation. O2 equilibria showed the lowest O2 affinity ever reported among vertebrate Mbs (P50 = 4.92 ± 0.29 mmHg, 25°C), a small overall heat of oxygenation (Δ H = −12.03 kcal/mol O2), and no effect of chloride, pH, or lactate. Although the reaction with 4,4′-dithiodipyridine (4-PDS) showed 1.3–1.9 accessible thiols per heme, the reaction of Mb with S-nitroso cysteine (Cys-NO) and S-nitrosoglutathione (GSNO) to generate Mb-SNO yielded ∼0.3–0.6 and ∼0.1 SNO/heme, respectively, suggesting S-nitrosation at only one cysteine (likely Cys10). At ∼60% S-nitrosation, trout Mb-SNO showed a higher O2 affinity (P50 = 2.23 ± 0.19 mmHg, 20°C) than unmodified Mb (3.36 ± 0.11 mmHg, 20°C). Total SNO levels measured by chemiluminescence in trout myocardial preparations decreased after hypoxia, but not significantly, indicating that transnitrosation reactions between thiols may occur in vivo. Our data reveal a novel, S-nitrosation-dependent allosteric mechanism in this low-affinity Mb that may contribute to targeted O2-linked SNO release in the hypoxic fish heart and be of importance in preserving cardiac function during intense exercise.