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 (ΔH = -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.
Helbo
Signe
S
Department of Biological Sciences, Aarhus University, Denmark.
Fago
Angela
A
D000494:Allosteric Regulation; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology; D023421:Models, Animal; D009211:Myoglobin / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D009574:Nitro Compounds / Q000378:metabolism; D015538:Nitrosation; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology; D026403:S-Nitrosothiols / Q000378:metabolism
D009211:Myoglobin; D009566:Nitrates; D009574:Nitro Compounds; D026403:S-Nitrosothiols; C116027:S-nitroglutathione; D009569:Nitric Oxide; C021315:S-nitrosocysteine; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen