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Rainbow trout myoglobin (Mb) is characterized by an unusually low affinity for oxygen, having a P(50) of 4.92±0.29 mm Hg at 25 °C which is the highest ever reported for any vertebrate Mb at the same temperature (Helbo and Fago, (2011) Am. J. Physiol. Regul. Integr. Comp. Physiol. 300, R101-R108). In order to gain insight into the structural factors of the heme pocket that may be important determinants for this atypical oxygen affinity, we have carried out an electronic absorption and resonance Raman characterization of the ferric and ferrous protein with and without exogenous ligands (O(2), CO, F(-)) and compared the results with those of other Mbs. While the ν(Fe-His) stretch appears at a frequency similar to other vertebrate Mbs, the resonance Raman frequencies of the Fe-ligand stretching modes reveal significant variations in the interaction of iron-bound ligands with distal residues. In particular, the spectroscopic characterization highlights two exceptional properties of rainbow trout Mb, a significantly higher level of reversed heme and reduced hydrogen bonding interactions between ligands and the distal HisE7 residue compared with other Mbs. The weakening of the hydrogen bond interaction is proposed to be the primary cause of the significantly reduced oxygen affinity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

The roles of nitric oxide synthase activity (NOS), nitrite and myoglobin (Mb) in the regulation of myocardial function during hypoxia were examined in trout and goldfish, a hypoxia-intolerant and hypoxia-tolerant species, respectively. We measured the effect of NOS inhibition, adrenaline and nitrite on the O(2) consumption rate and isometric twitch force development in electrically paced ventricular preparations during hypoxia, and measured O(2) affinity and nitrite reductase activity of the purified heart Mbs of both species. Upon hypoxia (9% O(2)), O(2) consumption and developed force decreased in both trout and goldfish myocardium, with trout showing a significant increase in the O(2) utilization efficiency, i.e. the ratio of twitch force to O(2) consumption, suggesting an increased anaerobic metabolism. NOS inhibition enhanced myocardial O(2) consumption and decreased efficiency, indicating that mitochondrial respiration is under a tone of NOS-produced NO. When trout myocardial twitch force and O(2) consumption are enhanced by adrenaline, this NO tone disappears. Consistent with its conversion to NO, nitrite reduced O(2) consumption and increased myocardial efficiency in trout but not in goldfish. Such a difference correlates with the lower O(2) affinity measured for trout Mb that would increase the fraction of deoxygenated heme available to catalyze the reduction of nitrite to NO. Whereas low-affinity trout Mb would favor O(2) diffusion within cardiomyocytes at high in vivo O(2) tensions, goldfish Mb having higher O(2) affinity and higher nitrite reductase activity appears better suited to facilitate O(2) diffusion and nitrite reduction in the heart during severe hypoxia, a condition particularly well tolerated by this species.