Search

Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxidative muscle but also in several other tissues, including capillary endothelial cells, whereas the other, Mb2, is a unique isoform specific to brain neurons. To help understand the functional roles of these diverged isoforms in the tolerance to severe hypoxia in the carp, we have compared their O(2) equilibria, carbon monoxide (CO) and O(2) binding kinetics, thiol S-nitrosation, nitrite reductase activities, and peroxidase activities. Mb1 has O(2) affinity and nitrite reductase activity comparable to most vertebrate muscle Mbs, consistent with established roles for Mbs in O(2) storage/delivery and in maintaining nitric oxide (NO) homeostasis during hypoxia. Both Mb1 and Mb2 can be S-nitrosated to similar extent, but without oxygenation-linked allosteric control. When compared with Mb1, Mb2 displays faster O(2) and CO kinetics, a lower O(2) affinity, and is slower at converting nitrite into NO. Mb2 is therefore unlikely to be primarily involved in either O(2) supply to mitochondria or the generation of NO from nitrite during hypoxia. However, Mb2 proved to be significantly faster at eliminating H(2)O(2,) a major in vivo reactive oxygen species (ROS), suggesting that this diverged Mb isoform may have a specific protective role against H(2)O(2) in the carp brain. This property might be of particular significance during reoxygenation following extended periods of hypoxia, when production of H(2)O(2) and other ROS is highest.

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.