Electrostatically Driven Second-Sphere Ligand Switch between High and Low Reorganization Energy Forms of Native Cytochrome <i>c</i>

We have employed a combination of protein film voltammetry, time-resolved vibrational spectroelectrochemistry and molecular dynamics simulations to evaluate the electron-transfer reorganization free energy (λ) of cytochrome <i>c</i> (Cyt) in electrostatic complexes that mimic some basic features of protein–protein and protein–lipid interactions. The results reveal the existence of two native-like conformations of Cyt that present significantly different λ values. Conversion from the high to the low λ forms is triggered by electrostatic interactions, and involves the rupture of a weak H-bond between first- (M80) and second-sphere (Y67) ligands of the heme iron, as a distinctive feature of the conformational switch. The two flexible Ω loops operate as transducers of the electrostatic signal. This fine-tuning effect is abolished in the Y67F Cyt mutant, which presents a λ value similar to the WT protein in electrostatic complexes. We propose that interactions of Cyt with the natural redox partner proteins activate a similar mechanism to minimize the reorganization energy of interprotein electron transfer

NO₂-AA decrease NOS expression in ANG II stimulated HK-2 cells.

<p>(A)Western blot of NOS expression in ANG II-treated HK-2 cells (1x10⁶ cells) pre-incubated 30 min with vehicle, Losartan or NO₂-AA was analyzed. (B)Densitometric analysis of the bands was performed and the % of the relative density of NOS to β-actin of the observed bands was plotted as the mean ± SD, n = 3. * express significant differences relative to either control or NO₂-AA treated cells (p<0.05).</p

NO₂-AA spares SDH and ATPase in kidney mitochondria.

<p>Isolated mitochondria, enriched with either AA or NO₂-AA as described in methods, were exposed to peroxynitriteand both SDH(A) and ATPase(B) specific activities determined. Results are representative of three independent experiments and correspond to the mean ± SD, n = 3. Controls of NO₂-AA addition in the absence of peroxynitrite addition were included for both complexes activities. * p<0.05 data relative to control mitochondria; # p<0.05 relative to peroxynitrite-treated mitochondria.</p

Schematic representation of mitochondrial dysfunction modulation by NO₂-AA in a cellular model of kidney cells activated by ANG II.

<p>On the left of the diagram, the stimulation with ANG II in HK-2 increases the O₂^.- production as well as inducible NOS expression and formation of peroxynitrite. This highly oxidizing molecule produces a decrease in the activities (red line) of the respiratory chain complex SDH (complex II) and ATPase (complex V) as well as increases oxidation and nitration of mitochondrial proteins. Therefore, HK-2 exposed to ANG II exhibited mitochondrial dysfunction. The right side of the diagram represent the modulation of cell damage by the nitroalkene. In the presence of NO₂-AA a reduction of ANG II-induced HK-2 damage is produced, with lower extents of O₂^.- production as well as lower levels of inducible NOS expression leading to a decrease in peroxynitrite formation. The activities of the respiratory chain complex are restored and NO₂-AA also prevents oxidation and nitration of mitochondrial proteins. In summary, NO₂-AA modulates ANG II mediated oxidative damage improving mitochondrial function.</p

Modulation of ANG II- mediated peroxynitrite production by NO₂-AA_.

<p>HK-2 cells (1x10⁶ cells) were treated as previously and exposed to 30 μM Fl-B for 3 h. The fluorescence of Fl-B was followed by flow cytometry. Representative histograms are shown for controls compared to the ANG II stimulated cells in the absence (A) or presence of 10 μM AA (B) or 10 μM NO₂-AA (C). (D) Quantitative analysis of the histograms was performed determining a M1 region that corresponded to the cell population that exhibits high fluorescence due to Fl-B oxidation by peroxynitrite. A control with the nitroalkene in the absence of stimulation with ANG II was included as a control. *, # express significant differences respect to control and ANG II-treated cells, respectively (p<0.05).</p

NO₂-AA improves mitochondrial function in ANG II stimulated HK-2 cells.

<p>(A)HK-2 cells (1x10⁶ cells, black line) were pre-incubated with vehicle, 5 or 10 μM NO₂-AA or 10 μM AA and then treated with 0.1 μM ANG II for 3 h.Oxygen consumption was recorded at 37°C in intact cells using high resolution respirometry (OROBOROS Oxygraph-2K). Arrows indicate steps in the titration regime, inducing the following respiratory states: Oligomycin, inhibition of ATP syntase; FCCP, maximal stimulation by uncoupling of oxidative phosphorylation, and antimycin A, inhibition of complex III. Respiratory control ratio (RCR) values(B), maximal respiratory rate (C) and spare respiratory capacity(D) were determined as explained in Methods section and plotted as the mean ± SEM, <i>n</i> = 4. * p<0.05 relative to control cells; # p<0.05 relative to ANG II-treated cells.</p

Site of action of dichloroacetate.

<p>DCA inhibits the mitochondrial enzyme PDH kinase, thereby maintaining the PDH complex in its unphosphorylated catalytically active state and facilitating the aerobic oxidation of glucose.</p

DCA prevents SOD1^G93A astrocyte neurotoxicity to motor neurons.

<p>Motor neuron survival 72 h after plating either on non Tg or SOD1^G93A-bearing astrocytes pretreated with DCA or vehicle as indicated. Data are expressed as percentage of non Tg control, mean ± SEM from four independent experiments. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control.</p

DCA improves mitochondrial function in the spinal cord of SOD1^G93A mice.

<p>Calculated respiratory control ratio (RCR) for spinal cord mitochondria from non Tg or SOD1^G93A mice treated with DCA or vehicle as indicated. Data are mean ± SEM from three independent experiments. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control.</p

DCA delays loss of grip strength and neuromuscular junction shrinkage in SOD1^G93A mice.

<p>A) Hind-limb grip strength records from non Tg or SOD1^G93A male mice treated with DCA or vehicle as indicated. DCA-treated non Tg animals did not show differences with control ones and data are not shown in order to simplify the graph. Data are mean ± SEM from 9 animals per group. *p<0.05, significantly different from SOD1^G93A control. B) ACh receptors labeled with TMR-BgTx in representative EDL neuromuscular junctions from non Tg (top), SOD1^G93A control (middle) or DCA-treated SOD1^G93A (bottom). Quantification of total TMR-BgTx-stained neuromuscular area in the different groups of animals. Data are expressed as percentage of non Tg control, mean ± SEM from 15–35 neuromuscular junctions from 2–4 animals per group. *p<0.05, significantly different from non Tg control. **p<0.05, significantly different from SOD1^G93A control. Scale bar: 30 µm.</p