It is now well established that CcO represents an important site of regulation of mitochondrial oxidative phosphorylation and ATP generation. A number of human diseases are linked to CcO deficiency or dysfunction [33
]. CcO activity is known to be regulated by an array of factors including allosteric modulators, protein modification, mutations in both mitochondrial and nuclear genes and physiological factors such as hormones [36
]. Although the functions of mitochondrial encoded subunits in mammalian organisms have been well established, relatively less is known about the role of nuclear encoded subunits in the assembly, structural integrity or activity of the mammalian complex [8
]. It is generally assumed that the nuclear subunits have a role in the regulation, or in binding physiological modulators [8
]. In this paper we provide a direct and unequivocal evidence for the role of CcO Vb in the assembly of the mouse CcO complex in the Vb mRNA silenced RAW 264.7 cells.
Previously we have shown that different tissues with different oxidative capacity such as liver and heart show varying levels of CcO Vb contents in relation their heme aa3 contents [17
]. Similarly, different compartments of the heart exhibiting different O2
loads also showed a difference in CcO Vb contents [17
]. More recently using a Langendorf perfusion system we showed that CcO Vb levels declined in rabbit hearts subjected to ischemia/reperfusion, in relation to their heme aa3 contents, suggesting preferential loss of some of the peripheral subunits from the holoenzyme complex which was also accompanied by a loss of CcO activity [18
]. Similarly, RAW 246.7 macrophages exposed to hypoxia also show selective decrease of subunits IVi1 and Vb and associated loss of CcO activity [18
]. Proteomic studies on mitochondria from cancer patients with reduced CcO activity also showed a reduction in CcO Vb subunit contents [41
In this study we sought direct evidence on the role of subunit Vb in CcO function/activity by generating RAW 264.7 macrophage cell line stably expressing siRNA to Vb mRNA. The cell line used in this study contains ~80% reduced Vb mRNA and subunit levels. Remarkably, VbKD cells showed markedly reduced holoenzyme complex, reduced bimolecular EPR signal suggesting an altered enzyme, a markedly increased level of subcomplexes, reduced heme aa3 content, disruption of ΔΨm, and vastly reduced CcO activity. Crystal structure of bovine CcO complex revealed that Vb subunit is a peripherally associated with CcO facing the matrix side of the complex [15
]. This raises the question on the role of CcO Vb either in the stability of the enzyme complex or in its assembly. Results on pulse-chase analysis of CcO in control and VbKD cells () clearly suggest the latter. Using transient siRNA transfection Campian et al., [24
] showed a reduction in CcO activity and ~20% reduction in CcO II. A marginal effect on the steady state level of other subunits in this study likely reflects a marginal depletion of Vb mRNA by the transient transfection method used [24
Interestingly, SDS PAGE analysis of both mitochondrial and whole cell extracts also showed a similar decrease in other subunits of CcO in VbKD cells. However, real-time quantitation of mRNA levels showed a small but significant increase in the steady state levels of these mRNAs. We believe that this increase is reminiscent of compensatory increases of mRNAs for mitochondrial energy transducing complexes and other proteins known to occur in cells with dysfunctional mitochondria [42
]. We also believe that reduced subunit level probably reflects rapid degradation of unassembled subunits in the matrix or in the cytoplasm. Extensive studies in yeast have shown that the assembly of CcO is a highly organized sequential process [25
]. Yeast strain with deleted subunit IV, the yeast homolog of mammalian subunit Vb, failed to assemble intact CcO complex [22
]. Similarly, mutations at the zinc finger domain of yeast CcO IV (homolog of mammalian Vb) severely affected CcO activity [21
]. These genetic data support our present observations on loss of CcO activity and loss of a number of other subunits in VbKD cells. Notably, knock down of Vb in our study caused reduction in the steady state levels of nuclear subunits IV and Va. In the yeast system however, knock down of CcO IV (homolog of mammalian Vb) only marginally reduced the steady state levels of nuclear subunits CcO Va/b (mammalian CcO IV) and CcO VI (mammalian CcO Va) [22
]. The reasons for this difference remain unclear.
Recently it was shown that a functional assembled CcO is necessary for the stability of complex I [13
]. Loss of CcO in knockout mice lacking CcO subunit 10, a CcO assembly factor, also resulted in the loss of complex I [45
]. The prevailing hypothesis is that presence of CcO in proper abundance is needed for the formation of respirasome [46
], the supercomplexes containing one or more copies of complexes I, III and IV. This arrangement is thought to increase the efficiency of electron transfer and minimizes the leakage of electrons that can potentially generate reactive oxygen radicals [46
]. In VbKD cells, however, despite a marked reduction in CcO complex and the supercomplex structures, we have observed no decrease in the content or activity of complex I. It is likely that CcO subunit 10, a known chaperone plays a direct or indirect role in the assembly of complex I as well. Further studies are therefore necessary to understand the role of CcO complex in the stability of complex I.
An important observation was the formation of mitochondrial ROS in VbKD cells. Mitochondrial electron transport chain is an important source of reactive oxygen species in respiring cells. Among the mitochondrial sites, complex I and III are considered as significant sources of ROS [49
]. ROS formation by complex I is thought to be due to the reverse flow of electrons under limiting substrate conditions. This is a common phenomenon during hypoxia where complex I is considered the major source of ROS. Also, ROS generated by this site may or may not depend on mitochondrial membrane potential depending on the substrate oxidized and the activities of the other energy transducing complexes [52
]. Complex III is another known site of superoxide formation where auto oxidation of accumulated semiubiquinone anion radical has been implicated and ROS production at this site depends on ΔΨm [52
]. To confirm the mitochondrial origin of ROS, we used a mitochondria targeted EPR spin trap, mitoDEPMPO [29
]. We found a significantly higher level of both hydroxyl and superoxide radicals in VbKD cells compared to control cells. Since CcO is not known to be a site of ROS production, the origin of these reactive species in VbKD cells needs to be established. It is possible that blocking of electron transport chain in these cells leads to the accumulation of reduced semiubiquinone, which in turn may be the cause of ROS. However, a disrupted ΔΨm in these cells is unlikely to support this reaction mechanism. Another possibility is the abnormal subcomplexes of CcO accumulated in the mitochondrial membrane in VbKD cells. Recently Khalimonchuk et al., [53
] showed in yeast that CcO subunit I-heme a, is a pro-oxidant intermediate formed during the assembly of cytochrome oxidase. Thus, assembly defective complexes of CcO in VbKD cells may be another source of reactive oxygen species.
In summary, in line with studies on subunit IVi1 and VIaH depletion reported recently [54
], our results show that the nuclear encoded peripheral subunit Vb plays a direct role in the assembly and activity of mammalian CcO complex. These results also suggest that subunit IVi1 and Vb, in particular may have a more direct role in the assembly or integrity of the complex rather than their previously predicted role in the regulation of enzyme activity. This is also the first demonstration of a structural role for a peripheral, non-integral membrane subunit of mammalian cytochrome oxidase.