The present study characterized the effect of the deletion of the Rieske iron-sulfur protein on CIII assembly and showed that the interdependence and supramolecular organization of OXPHOS complexes are associated with the increased levels of ROS.
The complete assembly process of CIII is still under investigation, but work in yeast suggested that it starts with the formation of 3 subassembly intermediates composed by subunits cytb/Qcr7/Qcr8, cytc1/Qcr6/Qcr9, and Core1/Core2. By an unknown assembly process, these three intermediates form a precomplex, pre-CIII, to which only the last two subunits (RISP and Qcr10) need to be inserted (70
). In yeast, three chaperones, Cbp3, Cbp4, and Bcs1, aid in the assembly process (16
). The addition of RISP is catalyzed by Bcs1 in an ATP-dependent manner, and it appears that the structural dimerization of the complex occurs before RISP is incorporated (16
). The mammalian CIII contains an additional subunit (UQCR9) that is derived by the proteolytic cleavage of the amino-terminal portion of RISP (5
). It is not clear when the cleavage of RISP to produce UQCR9 occurs, but presumably it is after its addition into the complex by BCS1L (human homolog of Bcs1). Interestingly, the Core1 and Core2 subunits display mitochondrion-processing peptidase activity (18
We did not detect major differences in the electrophoretic mobility of the fully assembled CIII and that of the one lacking RISP. Therefore, we have confirmed that in mammals, the dimerization and the formation of the pre-CIII assembly intermediate take place prior to RISP addition. Moreover, the pre-CIII appears to be structurally unstable, as we observed low levels of pre-CIII and subassembly intermediates/degradation products in the RISP KO clones. These results are in agreement with observations made in Bcs1L patients, where little incorporation of RISP into the pre-CIII occurs (25
The absence of RISP affected not only the levels of pre-CIII but also the levels of CI, CIV, and supercomplexes. Several possibilities that could contribute to the interdependence of respiratory complexes include the existence of specific threshold levels of respiratory complexes (17
), specific subunits and lipid interactions (47
), or as we showed in this study, increased levels of ROS.
Mitochondria, in particular the electron transport chain (ETC), are major generators of ROS (reviewed in reference 41
). The established sites for mitochondrial ROS production are CI, CII, and CIII. Complexes I and II produce superoxide within the mitochondrial matrix, whereas CIII releases the radicals into either the matrix or the mitochondrial intermembrane space (30
Analysis of RISP KO clones showed increased levels of ROS associated with destabilization of CI and supercomplexes. Complex I instability has been shown previously in both human and mouse cell lines with mutations in the cytochrome b
gene that impeded the assembly of CIII (1
). Studies in NDUFS4 KO mice showed that CI was stabilized by the formation of supercomplexes containing CIII in the absence of the CI subunit NDUFS4 (9
Our results suggest that CI stability is affected by factors other than the “physical support” conferred by CIII to assemble into supercomplex structures. Here, we show that the presence of RISP per se
is not required for CI stability, since the exposure of RISP KO fibroblasts to hypoxia and, presumably, low ROS was sufficient to increase CI to the levels observed in control cells. This indicates that the partial assembly of CIII into the pre-CIII in the RISP KO cells was enough to stabilize CI and supercomplex formation. Unfortunately, the previous studies on CI stability mentioned above did not investigate the effect of increased free radicals in stability/assembly (1
To test whether increased levels of ROS affected OXPHOS complex stability, we exposed control cells to different OXPHOS inhibitors known to increase free radicals. Studies in submitochondrial particles suggested that rotenone inhibits the binding of coenzyme Q to its reduction site in CI, permitting the release of electrons directly to oxygen at the N2 center and forming superoxide radicals (24
). The generation of free radicals by CIII is intimately linked to its catalytic mechanism. During the Q cycle, ubiquinol is oxidized and two electrons enter CIII in a bifurcate fashion. According to this model, inhibition of CIII with antimycin A and myxothiazol, which bind to center N or center P, respectively, can generate superoxide anions. Myxothiazol binds close to heme bL
and does not interfere with ubiquinol binding; in this way, ubiquinol electrons can access RISP but not heme bH
, allowing for the formation of superoxide (61
). Among the drugs that we used, CIII and CV inhibitors known to be ROS generators displayed the most detrimental effect on CI and supercomplex stability. Acin-Perez et al. (1
) showed that antimycin A decreased the levels of CI in mouse control cells derived from the L929 line (subcutaneous aerolar and adipose tissue) (1
). However, the decrease that they observed in CI levels was not as severe as the one we observed in this study. This difference could be attributed to variations in cell type or drug treatment time. It is possible that during their long treatment period with the drug (2 weeks), cells in culture adapted to the inhibition or that the “less fit” cells were selected against in growing cultures.
RISP is thought to be required for the production of reactive oxygen species by CIII, indicating that the increased levels of ROS observed in our RISP KO fibroblasts are probably generated by other sources (i.e., CI or CII). Along these lines, Hinson et al. (36
) showed that the levels of free radicals produced by mitochondria from patients with Björnstad syndrome (Bcs1L mutations) and other CIII deficiencies were increased by 50 to 80%. The authors determined that the increased levels of H2
detected in the patients were produced by CI and not by CIII (36
). More recently, Moran et al. confirmed increased levels of H2
and upregulation of ROS-scavenging enzymes in fibroblasts derived from patients with different mutations in the BCS1L gene. Remarkably, the levels of ROS in these patients correlated with the severity of the disease (49
). These results support our findings of increased ROS in the RISP KO cells.
The production of free radicals under hypoxia is still controversial. In this paradox, researchers supporting hypoxia-induced ROS contend that at low oxygen levels, the flow of electrons through the electron transport chain slows down, increasing the likelihood for the electrons to escape and produce free radicals. In contrast, other reports showed decreased ROS during hypoxia (23
). This controversy might rely on the nature of the free radical species measured. As pointed out by Poyton's group, not only reactive oxygen species but also reactive nitrogen species (RNS) can be generated during hypoxia (10
). Low levels of both ROS and RNS act as signaling molecules during physiological conditions, and only excessive amounts, usually generated during a pathological state, produce oxidative stress (34
). A potential reconciliation of this controversy came from the studies measuring the oxidation state of redox-sensitive GFP (roGFP) probes in different cellular compartments. Waypa et al. (66
) found that hypoxia increased the oxidation of roGFP in the cytosol and intermembrane space, whereas it decreased oxidation in the mitochondrial matrix (66
). Decreased mitochondrial matrix ROS supports our observations of increased OXPHOS complex stability/assembly during hypoxia.
It has been proposed that CIII is the mitochondrial oxygen sensor that regulates cellular responses during hypoxia and that the ROS generated by CIII is responsible for stabilizing the transcription factor HIF1-α that initiates the hypoxic signaling cascade (reviewed in reference 11
). This hypothesis is based in numerous studies performed in different cell types, including rho0
cells (cells devoid of mitochondrial DNA [mtDNA]), cytochrome b
and cytochrome c
mutants, specific OXPHOS inhibitors, mitochondrially targeted antioxidants, and knockdown studies (4
). Of particular interest are the studies on the effect of RISP knockdown on HIF1-α stability. Brunelle et al. (6
) showed that transient transfection of two RISP small interfering RNA sequences led to a reduction in the stabilization of HIF1-α in Hek293 cells exposed to hypoxia (1.5% O2
) for 2 h (6
). Stable knockdown of RISP in 143B cells showed similar results, and when exogenous H2
was added, hypoxic HIF1-α stabilization was restored, suggesting that ROS is required for stability of the transcription factor (33
). Hypoxic abrogation of HIF1-α stabilization upon knockdown of RISP (15
) and the role of ROS (46
) have been reported in other cell types also. Interestingly, recent studies implicate another CIII subunit (UQCRB) in the oxygen-sensing role during hypoxia (37
The use of MitoQ, a mitochondrially targeted antioxidant, supported the hypothesis that mitochondrial ROS was required for hypoxic signaling. Control and cytochrome b
mutant cybrids pretreated with MitoQ prior to hypoxic exposure failed to stabilize HIF1-α (4
). This concept has been recently challenged by Chua et al. (14
). The authors observed that pharmacological inhibition of the electron transport chain decreased the HIF1-α half-life under hypoxia to the same extent independent of the complex inhibited and that there was no increase in ROS production during hypoxia (14
). The authors proposed that, rather than requiring ROS produced by CIII, HIF1-α stability during hypoxia is related to the intracellular concentrations of oxygen, which are determined by the rate of mitochondrial respiration.
Similar to results of the RISP knockdown studies described above, we were able to detect a marked decrease in the levels of HIF1-α after exposing our RISP KO cells to hypoxia (1% O2
) for 4 h. However, after 24 h of hypoxia, the levels of HIF1-α in KO cells increased, reaching levels that were even higher than the ones in control fibroblasts. Moreover, reducing mitochondrial ROS with different concentrations of MitoQ did not have a significant effect on HIF1-α stability. This observation suggests that HIF1-α stability in the RISP KO cells during hypoxia may be independent of ROS production, as has been suggested (14
). The RISP KO cells had increased levels of superoxide, and antioxidant defenses were upregulated. Presumably, in our cells, ROS is generated by means other than CIII. However, we did not observe an increase in hydrogen peroxide during hypoxia.
We were surprised to find that hypoxia also increased the stability/assembly of CI, CIV, and supercomplexes. Although the results of the experiments described above do not rule out the participation of HIF1-α in this process, they showed that ROS is an active mediator of complex/supercomplex stability. Interestingly, there are two forms of CI: one form, called the A-form, is catalytically active, and the other, called the D-form, is catalytically inactive (65
). Deactivation occurs when all reactive redox centers of CI are in the reduced state. During hypoxia, a deactivation of CI was observed in epithelial kidney cells (27
). The authors proposed that the deactivation of CI could be a protective mechanism for a potential burst of free radicals during reoxygenation (27
). Unfortunately, they did not investigate the stability of the CI and supercomplexes during hypoxia.
The fact that we observed that conditions producing high levels of free radicals (antimycin A, myxothiazol, and oligomycin) led to CI instability and affected the levels of supercomplexes and, conversely, that conditions of lower oxygen and, presumably, lower levels of free radicals preserved supercomplexes even in OXPHOS-defective cells prompts us to propose a possible model for regulation of OXPHOS complex interactions into supercomplexes. illustrates this model: in wild-type cells, OXPHOS complexes are able to form stable supercomplexes. Alterations in OXPHOS function can produce increased free radicals, which are potentially dangerous. To avoid this, CI is degraded and supercomplexes disassembled. Under conditions of low oxygen or increased superoxide scavengers (SOD2 or MnTBAP), less ROS is produced, resulting in the restoration of a safe environment for the respiratory complexes and supercomplexes to reassemble. In addition, supercomplex stability/assembly could be further regulated by the expression of hypoxia-specific isoforms of subunits of the respiratory chain and could be mediated by HIF1-α.
Fig 11 Model of hypoxic-induced stability of OXPHOS complexes and supercomplexes. The mitochondrial OXPHOS complexes are able to associate, forming stable supercomplexes in wild-type (WT) cells. Defects in OXPHOS function, such as the absence of RISP, can cause (more ...)
Our results also addressed a previously puzzling observation. Mouse tissues defective in CIV (21
) or CIII (F. Diaz, unpublished observations) did not show a decrease in CI, as consistently observed in cultured cells. However, the oxygen concentration in tissues is 3 to 6%, which is markedly lower than the 21% concentration observed in cultured cells. Therefore, we feel compelled to speculate that increases in tissue oxygenation (physiological or pathological) may lead to a signaling pathway that controls the levels of CI and supercomplexes to avoid further exacerbation of the oxidative stress, as postulated in our model.
In conclusion, our data suggest that localized increases in ROS levels at the mitochondrial inner membrane affect the assembly/stability of CI and supercomplexes. CI appears to be particularly sensitive to this oxidant environment effect. This system may be physiologically relevant for the control of respiration and ROS levels.