Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I

Abstract

Aims: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio.

Results: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I.

Innovation: Dissociation of supercomplexes may link oxidative stress and energy failure in a vicious circle.

Conclusion: Our findings support a central role of mitochondrial supramolecular structure in the development of the aging process and in the etiology and pathogenesis of most major chronic diseases.

FIG. 1.

Supercomplex disassembling in bovine heart mitochondria (BHM). Respiratory supercomplexes and complexes from (A) digitonin-solubilized BHM with a detergent to protein ratio (w:w) of 8 and from (B) DDM-solubilized BHM with a detergent to protein ratio (w:w) of 2.6 were resolved by western blotting after 2D-BN/SDS-PAGE. Arrows point to monomeric Complex I. Monoclonal antibodies specific for single subunits of each OXPHOS complex are as follows: NDUFB8 (20 kDa) and NDUFA9 (39 kDa) of Complex I, SDHB (30 kDa) of Complex II, Core protein 2 (47 kDa) of Complex III, and COX-I (57 kDa, apparent 35 kDa) of Complex IV and alfa subunit (53 kDa) of ATP synthase. The images shown in the picture were obtained in camera by double overlaying exposures (not postproduction computer-graphic overlay) to the antibody against the NDUFA9-subunit and, in sequence, to a mixture of the remaining antibodies listed above. DDM, dodecyl-β-D-maltoside; OXPHOS, oxidative phosphorylation system.

FIG. 2.

Functional analysis of supercomplex I₁III₂ and Complex I in detergent-solubilized BHM. Detergent dependence of (A) NADH-ubiquinone oxidoreductase activity and (B) NADH-cytochrome c oxidoreductase activity. Activity rates are expressed in μmoles of NADH·min⁻¹·mg protein⁻¹. (C) The production of hydrogen peroxide was measured in the presence of 1.8 μM mucidin (dashed bars) and 1.8 μM mucidin plus 4 μM Rotenone (solid bars). Values are expressed as relative fluorescence units (RFU) by DCF normalized against Complex I activity and shown as percentage values of the reference sample (i.e., control BHM in the presence of mucidin only). The corresponding percentage values of RFU·mg protein⁻¹ in the presence of mucidin plus rotenone are 121±20 and 158±61, respectively in control and in DDM-treated samples. DDM sample treated with 2.6 g dodecylmaltoside/g of protein; n indicates the number of independent samples. Data are given as mean±S.D. p-values were calculated using the Student's t-test. DCF, 2′,7′-dichlorofluorescein.

FIG. 3.

Supramolecular organization of respiratory Complex I and Complex III in R4B 1:1 and R4B 1:30 proteoliposomes. (A) R4B 1:1 and (B) R4B 1:30 samples were separated by 2D BN/SDS-PAGE after solubilization with digitonin at a detergent to protein ratio of 8 (w:w) and resolved by western blotting followed by immunodetection using monoclonal antibodies specific for single respiratory subunits. The images shown in the picture were obtained in camera by double overlaying exposures (not postproduction computer-graphic overlay) to the antibody against the NDUFA9 (39 kDa) subunit of Complex I and, in sequence, to the antibody against the Rieske protein (22 kDa) of Complex III. Arrows point to monomeric Complex I. The upper panel of the figures schematically shows the position of supercomplex I₁III₂ (SC), Complex I (CI) and dimeric Complex III (CIII) in the 1D BN-gel prior to second dimension electrophoresis (2D SDS-PAGE). NADH-cytochrome c and NADH-ubiquinone oxidoreductase activities are expressed in nmoles of NADH·min⁻¹·mg protein⁻¹. n indicates the number of independent samples. Data are given as mean±S.D. Numbers followed by asterisks are significantly different according to the Student's t-test (*p<0.05). R4B, mitochondrial fraction enriched in Complex I and Complex III.

FIG. 4.

ROS production mediated by Complex I in R4B 1:1 and R4B 1:30 proteoliposomes. (A) The rate of superoxide formation is determined as the superoxide dismutase (SOD)-sensitive rate of acetylated cytochrome c reduction in the presence of 1.8 μM mucidin and 4 μM rotenone. The corresponding rates of SOD-insensitive activity are: 1.9±1.7 and 1.7±1.0 nmol·min⁻¹·mg protein⁻¹, respectively in the 1:1 versus 1:30 samples. (B) The production of hydrogen peroxide was measured in the presence of 1.8 μM mucidin (dashed bars) and 1.8 μM mucidin plus 4 μM Rotenone (solid bars). Values are expressed as relative fluorescence units (RFU) by DCF normalized against Complex I activity and shown as percentage values of the reference sample (i.e., R4B 1:1 in the presence of mucidin only). n indicates the number of independent samples. Data are given as mean±S.D. p-values were calculated using the Student's t-test. ROS, reactive oxygen species.

FIG. 5.

Disassembling of supercomplex I₁III₂ in R4B1:1 proteoliposomes after detergent solubilization. (A) Respiratory complexes were separated by 2D BN/SDS-PAGE after solubilization with DDM with a detergent to protein ratio (w:w) of 4.3 and resolved by western blotting using monoclonal antibodies specific for single respiratory subunits. The images shown in the picture were obtained in camera by double overlaying exposures (not postproduction computer-graphic overlay) to the antibody against the NDUFA9 (39 kDa) subunit of Complex I and, in sequence, to the antibody against the Rieske protein (22 kDa) of Complex III. The arrow points to monomeric Complex I. The upper panel of the figure schematically shows the position of supercomplex I₁III₂ (SC), Complex I (CI), and dimeric Complex III (CIII) in the 1D BN-gel prior to second dimension electrophoresis (2D SDS-PAGE). The detergent dependence of (B) NADH-ubiquinone oxidoreductase and (C) NADH-cytochrome c oxidoreductase activities in the DDM-treated samples was estimated by comparison to the corresponding values in R4B 1:1 samples without DDM (control). (D) The production of hydrogen peroxide was measured in the presence of 1.8 μM mucidin (dashed bars) and 1.8 μM mucidin plus 4 μM Rotenone (solid bars). Values are expressed as relative fluorescence units (RFU) by DCF normalized against Complex I activity and shown as percentage values of the reference sample (i.e., control R4B 1:1 in the presence of mucidin only). DDM sample treated with 4.3 g dodecylmaltoside/g of protein. NADH-ubiquinone oxidoreductase and NADH-cytochrome c oxidoreductase activities are expressed in μmoles of NADH·min⁻¹·mg protein⁻¹. n shows the number of independent samples. Data are given as mean±S.D. p-values were calculated using the Student's t-test.

FIG. 6.

Production of ROS by mitochondrial Complex I in different situations where supercomplexes are maintained or disassembled. The percent value of ROS production measured in all the samples listed in Table 1 is plotted in the graph against the corresponding ratio of free Complex I versus total Complex I. The statistical analysis of the data using the Pearson's parametric test indicates a positive correlation (r=0.884, p<0.05) between ROS generation and relative amount of free Complex I in the R4B and in the SMP samples (black symbols). The BHM samples (gray symbols) were not included in the correlation analysis because the existence of endogenous antioxidant systems operating to reduce ROS levels in the mitochondria might have counteracted the dramatic effects of the complete dissociation of Complex I, thus leading to a two-fold only increase of the measured ROS production. SMP, submitochondrial particles.