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

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

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

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

Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme <i>b</i>. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A₀) and closed (A₁) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme <i>b</i> forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A₀ conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme <i>b</i> at excess of hydrogen peroxide

Influence of CyaY on the enzymatic activity of IscS.

<p><b>A</b>) Dosage of the alanine formed at different times. Each measurement is reported twice and corresponds to 5 and 20 min after starting the reaction. S, SU and SUC indicate different mixtures of IscS, IscU and CyaY. <b>B</b>) Kinetics of cysteine consumption with concomitant alanine formation. Peak intensities of the b-protons of alanine (left) and cysteine are reported as a function of time. <b>C</b>) Same as in B) but plotting the results of experiments recorded in the presence of different mixtures of IscS, IscU and CyaY. The two curves in the presence of IscU (which superpose perfectly) correspond to two independent kinetics performed with 12 hours interval to check for reproducibility.</p

Mössbauer spectra of the FeS cluster assembly reaction.

<p>The spectra were recorded in the absence (left panel, spectra A (top) – D (bottom)) and in the presence (right panel, spectra E (top) – H (bottom)) of CyaY. The spectra were recorded at 4.2 K and with a magnetic field of 60 mT applied parallel to the γ ray.</p

Time dependence of FeS cluster formation deduced from the Mössbauer experiments.

<p>The individual contributions of the different complexes are estimated at each time point (5, 15 and 30 min) in the absence (left) and in the presence (right) of CyaY.</p