Abstract
The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O2 affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O2 affinity (P 50 = 2.7 torr) that was increased by S-nitrosation (P 50 = 1.7 torr), whereas in tuna Mb, O2 affinity (P 50 = 0.9 torr) was independent of S-nitrosation. O2 dissociation rates (k off) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O2 affinity by raising the O2 association rate (k on). Taken together, these results indicate that O2-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O2 entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.
Highlights
S-nitrosation is a widespread and reversible post-translational protein modification in which formally a nitric oxide (NO) molecule is covalently bound to a reactive thiol on a specific cysteine (Cys) yielding S-nitrosothiol (SNO)
We examine whether Mbs from two other fish species, the yellowfin tuna and the Atlantic salmon, containing one (Cys10) and two Cys residues (Cys10 and Cys107), respectively (Figure 1), undergo changes in O2 affinity upon S-nitrosation similar to those described in rainbow trout Mb, containing Cys10 and Cys107 (Figure 1)
This finding is consistent with their amino acid sequences, which show that yellowfin tuna Mb has one Cys (Cys10), Atlantic salmon Mb has two Cys (Cys10 and Cys108) and human Mb has one Cys (Cys110) (Figure 1)
Summary
S-nitrosation is a widespread and reversible post-translational protein modification in which formally a nitric oxide (NO) molecule is covalently bound to a reactive thiol on a specific cysteine (Cys) yielding S-nitrosothiol (SNO). Human hemoglobin (Hb), the O2 carrier of the blood, was one of the first proteins discovered to be S-nitrosated at Cysb to generate Hb-SNO [2,3] This protein modification increases the heme oxygen (O2) affinity (P50, the O2 tension at halfsaturation) [4,5] by shifting the allosteric equilibrium between two alternative tetrameric quaternary structures of the Hb. the shift in the opposite direction by a decrease in O2 levels destabilizes the S-NO bond causing the release of the NO moiety, which may induce vasodilation and increase local blood flow in hypoxic tissues [2,3,6]. The extent of NO scavenging by deoxy heme appears to be limited in the hypoxic myocardium of trout, suggesting a role of endogenously produced NO in the regulation of myocardial O2 consumption [12]
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