Redox cycle of myoglobin.

<p>Ferric Mb reacts with hydrogen peroxide to give the long-lived oxoferryl species and a transient protein radical (tryptophanyl radical), directly formed or resulting from electron transfer from the protein to the porphyrin π cation radical (steps <i>a</i> and <i>b</i>). The long-lived oxoferryl Mb can recycle to the resting form by two electron reduction (step c). Tryptophanyl free radical could react with molecular oxygen to form a peroxyl-derived free radical (step <i>d</i>). The protein radical may lead to the Mb degradation (bleaching) as shown in step <i>e</i>.</p

Maldi-ToF mass spectra of Mb incubated at different conditions: absence of H₂O₂.

<p>(A), the products of a 10 min reaction with H₂O₂, in the presence of 10 mM DBNBS obtained in the absence (B) and in the presence (C) of TOP. Reactions were carried out in 20 mM tris buffer pH 7.4.</p

Mechanism of TOP oxidation by hydrogen peroxide.

<p>Mechanism of TOP oxidation by hydrogen peroxide.</p

Production of TOP thiyl radical by Mb.

<p>The Mb-catalized one-electron oxidation of TOP is expected to generate thiyl free radical and the fate of this free radical is proposed according to reference 55.</p

Experimental EPR spectra of tryptophanyl DBNBS adducts obtained from the reaction mixture containing Mb and H₂O₂.

<p>A) Absence of TOP. Line a and b correspond, respectively, to the EPR spectra of 20 mM DBNBS adduct obtained immediately and 10 min after the addition of 50 µM H₂O₂ to 5 µM Fe³⁺Mb solution. The light gray line, overlapped on line a, corresponds to the spectrum simulated by NLS software, B) Presence of 20 µM TOP: Lines a, b, c and d correspond to the experimental EPR spectrum, simulated composite EPR spectrum, and the simulated rigid and free rotating components, respectively. The spectrum was obtained immediately after the addition of 50 µM H₂O₂ in 5 µM Fe³⁺Mb solution. When present, TOP was previously treated with 1 mM TCEP. EPR conditions were: microwave frequency = 9.5077 GHz, central field, 340 mT, scanning field, 16 mT, number of points, 1024, modulation amplitude, 0.05 mT, gain, 5.0x10⁵, temperature, 293 K, time constant, 0.128 s, scan time, 300 s, microwave power, 20 mW. The reaction was performed in buffer Tris 20 mM previously treated with Chelex-100 ®, pH 7.4, at 25° C.</p

Proposed mechanism of TOP participation in the redox cycling of myoglobin.

<p>In the presence of TOP, steps <i>a</i> and <i>b</i> also occurs and the Mb “Compound I” is recycled to the resting form by using TOP thiol groups as a reducing agent (steps <i>c</i> and <i>d</i>).</p

Catalytic cycle of Mb.

<p>A) Absence of TOP. Electronic absorption spectra of 0.5 µM Mb before (thin solid line),10 min (gray line) and 120 min (thick solid line) after the addition of 5 µM H₂O₂. B) Presence of TOP. Electronic absorption spectra of 0.5 µM Mb before (thin solid line),10 min (gray line) and 35 min (thick solid line) after the addition of 5 µM H₂O₂. C) Effect of TOP concentration on the rate of Mb Compound II recycling to the resting form. After normalization of the spectra of Mb Compound II and recycled Mb by the maximal intensity of Soret band, the rate of oxoferryl Mb recycling to the resting state was calculated by the difference of intensity at 408 nm and the normalized spectra of the recycled Mb. The delta normalized absorbance at 408 nm was divided by the time of recycling and plotted as a function of TOP concentration. Different TOP concentrations were added 10 min after hydrogen peroxide addition (maximal Soret band red shift). Immediately after the formation of Compound II, 5 µM of TOP was added to the medium. These results are representative of a set of two independent experiments with standard deviation of 15%. The reaction was carried out at 37 °C, in 20 mM Tris buffer pH 7.4 treated with Chelex-100®. </p

SH content and aggregates of TOP treated with 10 mM (100% reduced cysteines) and 1 mM (40% reduced cysteines) TCEP.

<p>A) WT) wild type TOP, A) + 10 mM TCEP, B) + 10 mM TCEP and 5 µM H₂O₂, C) + 10 mM TCEP and 0.5 µM Mb, D) + 10 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E) + 10 mM TCEP and 0.5 µM HRP, F) + 10 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂, A') + 1 mM TCEP, B') + 1 mM TCEP and 5 µM H₂O₂, C') + 1 mM TCEP and 0.5 µM Mb, D') + 1 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E') + 1 mM TCEP and 0.5 µM HRP, F') + 1 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂. B) SDS-PAGE of TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 100% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. C) SDS-PAGE of 40%-reduced TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 40% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. D) This panel shows the large amount of aggregates in aged TOP. The reaction mixtures were incubated for 2 hours in buffer Tris 20 mM Chelex-100 ®, pH 7.4, at 37° and contained 5 µM TOP. The SDS-PAGE of 100% reduced TOP was done with 10 μM TOP.</p

Interatoma of rat Cygb with hydrogen peroxide.

<p>The network shows, in each node, a protein predicted to have functional links with Cygb and hydrogen peroxide. Inside the figure the abbreviations are SOD1 (superoxide dismutase [Cu-Zn]), Hmox2 (heme oxygenase 2 [HO-2]), Mb (myoglobin), Mpo (myeloperoxidase), cat (catalase), Cygb (cytoglobin), Prdx1 (peroxyredoxin-1), Prdx5 (peroxyredoxin-5) and Srxn1 (Ab2-390). In the figure light green, cyan and magenta lines correspond, respectively, to textmining, databases and experiments supporting the relationship among the proteins and hydrogen peroxide.</p

Changes in the EA spectrum of Cygb during the reaction with <i>t</i>-BuOOH.

<p>A) Bleaching of Soret and Q bands of EA spectra of Cygb in the course of the reaction with <i>t</i>-BuOOH. The black line represents the EA spectrum of resting Cygb, red, green and blue lines corresponds to the spectra obtained at 30, 60 and 200 s after addition of <i>t</i>-BuOOH and indicated by the arrows. B) Normalized spectra of Cygb resting form and 200 s after <i>t</i>-BuOOH addition. C) Differential spectra of Cygb obtained 30 and 200 s after the addition of <i>t</i>-BuOOH. The experiments of EA spectroscopy were performed using 65 μmol.L^-1 Cygb and 0.1 cm optical length. When present, the concentration of peroxides was 650 μmol.L^-1. These results are representative of three independent replicates.</p