Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

<div><p>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 <i>in vitro</i> 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 (<i>P</i>₅₀ = 2.7 torr) that was increased by S-nitrosation (<i>P</i>₅₀ = 1.7 torr), whereas in tuna Mb, O₂ affinity (<i>P</i>₅₀ = 0.9 torr) was independent of S-nitrosation. O₂ dissociation rates (<i>k</i>_off) of trout and salmon Mbs were not altered when Cys were in the SNO or <i>N</i>-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O₂ affinity by raising the O₂ association rate (<i>k</i>_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 <i>in vivo</i> is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.</p></div

Salmon and tuna Mbs have faster reacting Cys than human Mb.

<p>Time course of the reaction of 4-PDS with accessible free thiols of salmon (black dots), tuna (white circles) and human Mb (grey circles) were measured in 50 mM Hepes, pH 7.2 at 20°C at a ratio of 4∶1 4-PDS/heme. Data fittings by double (salmon) or single (tuna and human) exponential equations are indicated.</p

S-nitrosation increases O₂ affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by <i>N</i>-ethylmaleimide.

<p>A) O₂ equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O₂ equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097012#pone.0097012-Helbo1" target="_blank">[9]</a>.</p

Amino acid sequence alignment of salmon, trout, tuna, carp Mb1 and Mb2 and human Mb shows variable number and position of Cys residues (highlighted).

<p>Mb sequences have been retrieved from Pub Med (Atlantic salmon: Atlantic salmon: GenBank, ACM09229.1, rainbow trout: GenBank, BAI45225.1, yellowfin tuna: GenBank: AAG02112.1, common carp Mb1: UniProtKB/Swiss-Prot, P02204.2, common carp Mb2: GenBank: ABC69306.1, human: NCBI Reference Sequence: NP_976311.1). The positions of the α-helices (A–C, E–H) are indicated and are based on the structure of yellowfin tuna Mb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097012#pone.0097012-Schreiter1" target="_blank">[18]</a>.</p

Measured O₂ affinities (<i>P</i>₅₀) and kinetic O₂ dissociation rates (<i>k</i>_off) (means ± SD; n = 3) and derived O₂ dissociation equilibrium constants (<i>K</i>_d) and apparent kinetic O₂ association rates (<i>k</i>_on) for myoglobins from yellowfin tuna, Atlantic salmon and rainbow trout in the native (Mb) and thiol-modified forms (Mb-SNO and Mb-NEM).

<p>Linearly extrapolated values at 1.0 SNO/heme ratio and 1.0 NEM/heme ratio are indicated. Apparent <i>k</i>_on values are reported for identical degrees of thiol modification. Experimental conditions: 20°C, pH 8.3.</p>1<p>0.6 SNO/heme, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097012#pone.0097012-Helbo1" target="_blank">[9]</a>. ²0.4 SNO/heme. ³0.5 SNO/heme. ⁴0.6 SNO/heme. ⁵0.75 NEM/heme.</p><p>*Significantly different from the unmodified form (<i>P</i><0.05), unpaired t-test.</p

Biotin switch analysis of trout hearts indicates changes in Mb-SNO levels following incubation of trout heart preparations in oxygenated and anoxic conditions.

<p>Three freshly isolated ventricle rings were prepared from a single heart and either directly frozen (control) or incubated for 45 minutes in the presence (50% O₂) or absence (100% N₂) of O₂. Following incubation, the rings were immediately frozen. Lysates were prepared from heart rings and analyzed by the biotin switch technique. Samples labeled in the presence of NEM-Biotin are marked as +, while those in which biotin was omitted (−) serve as individual controls. Image shows rings isolated from two separate hearts. Biotin-labeled Mb was quantified by densitometry and then normalized to the total protein content. The mean ± standard error Mb-SNO level (n = 5) is given below the figure.</p

Scatter plot of circulating levels of serum heat shock protein 70 (Hsp 70) in the different groups of women with median and interquartile range.

<p>Group 1 (non-pregnant women, n = 14), group 2 (normal pregnant women, n = 32), group 3 (threatened miscarriage patients with a live birth outcome, n = 21) and group 4 (threatened miscarriage patients with a subsequent miscarriage, n = 19). General linear analysis of variance was carried out to study the statistical significance between the groups with posthoc tests. NS = not significant.</p

Scatter plot of circulating levels of serum soluble Endoglin (sEndog) in the different groups of women with median and interquartile range.

<p>Group 1 (non-pregnant women, n = 14), group 2 (normal pregnant women, women, n = 32), group 3 (threatened miscarriage patients with a live birth outcome, n = 21) and group 4 (threatened miscarriage patients with a subsequent miscarriage, n = 19). General linear analysis of variance was carried out to study the statistical significance between the groups with posthoc tests. P<0.001 = ***.</p

Demographic details of the participants in the study given as median and interquartile range (25^th, 75^th percentiles).

<p>Demographic details of the participants in the study given as median and interquartile range (25^th, 75^th percentiles).</p

Scatter plot of circulating levels of serum placental growth factor (PlGF) in the different groups of women with median and interquartile range.

<p>Group 1 (non-pregnant women, n = 14), group 2 (normal pregnant women, n = 32), group 3 (threatened miscarriage patients with a live birth outcome, n = 21) and group 4 (threatened miscarriage patients with a subsequent miscarriage, n = 19). General linear analysis of variance was carried out to study the statistical significance between the groups with posthoc tests. P<0.001 = ***, P<0.01 = **.</p