Allosteric modulation by S-nitrosation in the low-O₂affinity myoglobin from rainbow trout

Abstract

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.

Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia

“…Under this model, βCys93 is oxidatively modified by NO to form an SNO in R-state (oxy) Hb, and the allosteric structural transition of the Hb tetramer to T-state upon deoxygenation promotes the graded release from βCys93 of SNO-based vasodilatory bioactivity (6). Interestingly, an O 2 -coupled allosteric mechanism also may operate in the generation and release of NO bioactivity from myoglobin in cold-blooded vertebrates (13). Additional findings show that the export of βCys93-derived NO bioactivity is based on an SNO cascade that involves the transfer of NO groups to RBC membrane proteins and to external sites that include small-molecular-weight thiols (10,(14)(15)(16)(17).…”

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles

“…Mb1 was purified from heart homogenates by ammonium sulfate fractionation (40 and 80%) followed by fast protein liquid chromatography (FPLC) gel filtration using a Tricorn Superdex 75 10/300 GL column (GEHealthcare) equilibrated with 50 mM Tris, 0.5 mM EDTA, 0.5 mg/ml DTT, and 0.15 M NaCl, pH 8.3, as described in detail elsewhere (32). Purity of Mb1 was assessed by SDS-PAGE (21).…”

“…A modified diffusion chamber technique described before (21,27,41) was used to measure O 2 equilibrium curves on 4-l Mb samples (ϳ50 M heme) in 0.1 M HEPES buffer as a function of temperature (18,20,25, and 32°C), pH (6.4, 7.4, and 8.3), and in the absence and presence of lactate (10 and 100 mM at pH 6.5 for Mb1 and at pH 7.4 for Mb2). Briefly, in this method, a watersaturated gas mixture of O 2 or air and ultrapure (99.998%) nitrogen gas (N2) created by Wösthoff (Bochum, Germany) gas mixing pumps was used to equilibrate a thin smear of Mb solution placed in a modified diffusion chamber equipped with a photomultiplier (model RCA 931-A) and an Eppendorf model 1100 M photometer coupled to a potentiometric linear recorder.…”

“…Furthermore, Mb may reduce the massive production of the reactive oxygen species (ROS) generated upon reoxygenation after prolonged hypoxia (2) by metabolizing a major endogenous ROS such as hydrogen peroxide (H 2 O 2 ), thereby protecting cells from oxidative damage subsequent to hypoxia (12). Furthermore, cysteine (Cys) residues on Mb may become S-nitrosated (i.e., covalently bound to a NO moiety as SNO) (26,33,39), which may allosterically regulate heme oxygenation, as found in rainbow trout Mb (21), and be able to further contribute to NO homeostasis under hypoxia. Although well described in mammals, these novel functions of Mb have so far only been examined to very little extent in fish.…”

Functional properties of myoglobins from five whale species with different diving capacities

“…Mb from BNW, HW, MW and HP was purified from muscle as previously described (Helbo and Fago, 2011). In brief, Mb was precipitated from muscle homogenates by ammonium sulphate fractionation (40 and 100%) followed by fast protein liquid chromatography gel filtration using a Tricorn Superdex 75 10/300 GL column (GE Healthcare, Broendby, Denmark) equilibrated with 50mmoll -1 Tris, 0.5mmoll -1 EDTA, 3mmoll -1 dithiothreitol (DTT), 0.15moll -1 NaCl, pH8.3 to separate Mb from contaminating Hb.…”