Assembly of cytochrome‐c oxidase in cultured human cells

Abstract

The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using twodimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.Keywords : cytochrome-c oxidase; assembly; respiratory chain ; cultured human cell.Cytochrome-c oxidase (COX) is the terminal enzyme of the in a stable intermediate complex made up solely of nuclear-encoded subunits [10,11]. respiratory chain located in the inner mitochondrial membrane. The enzyme complex catalyses the oxidation of cytochrome c Basic questions regarding the assembly of COX remain to be resolved. What is the order of assembly of the subunits? How by molecular oxygen; the energy released in this reaction is used to translocate protons across the inner mitochondrial membrane. is assembly regulated and what are the rate-limiting steps? Answers to these questions are not only important to understand The proton gradient resulting from the activity of the different respiratory-chain complexes is used to drive ATP synthesis. the function and regulation of the enzyme, but they will also provide information on causes of COX deficiency in patients. Mammalian COX contains 13 subunits, three of which (COX I, II and III) are encoded by the mitochondrial genome, while the Most investigations of the assembly pathway of COX employed metabolic labeling followed by immunoprecipitation [8Ϫ remaining ten subunits (COX IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc and VIII) are nuclear-encoded [1Ϫ5].10, 12]. This approach may give erroneous results because there are differences in epitope availability in partially assembled The recent resolution of bovine heart COX by X-ray analysis [6, 7] not only revealed the exact topology of its subunits, but complexes or because there is selective loss of subunits during immunoprecipitation. We have used two-dimensional electroalso assists studies aimed at clarifying the reaction mechanism of the enzyme. Moreover, the quarternary structure of the com-phoresis in combination with translational inhibitors and metabolic labeling to analyze COX assembly. Two assembly intermeplex puts certain structural constraints on models regarding the assembly pathway of COX. To resolve the assembly process of diates could be detected. the enzyme, however, additional experimental approaches are required. Previous studies of the assembly pathway of COX have shown that pools of unassembled subunits exist and that EXPERIMENTAL PR...

Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells

“…7,8,11,12 One of the key mitochondrial proteins is COX, the fourth complex and the rate-limiting step of the ETC, where it expends the majority of oxygen for reduction by cytochrome c to produce water, and as a result generates adenosine triphosphate (ATP) via oxidative phosphorylation. 2,13 Given the antiapoptotic nature of Bcl-2 and the functional significance of COX in mitochondrial respiration, here we investigated the role of Bcl-2 in mitochondrial respiration with relation to COX and the subsequent downstream effects. We present evidence that cancer cells overexpressing Bcl-2 have greater basal COX activity and oxygen consumption as compared to mock-transfected cells.…”

A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

“…COX7A has been considered as a late-assembling subunit similar to COX6A ref. 29, and in vitro translated COX7A was first incorporated into complex IV and supercomplex III þ IV based on in vitro import and assembly assays 30 . Thus, it is likely that COX7A has a role in the late steps of holo-complex IV formation, as well as in the assembly of III and IV, and is located in the periphery of complex IV.…”

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

“…COX assembly is a linear process in which at least three subassemblies, S1, S2, and S3, can be detected, which probably represent the rate-limiting steps of the process (5). In this model, originally proposed by Nijtmans et al (5), S1 is formed exclusively by COX1, and the addition of subunits COX4 and COX5a to S1 results in the progression to S2.…”

“…In the current model of human COX assembly, the first subassembly (S1) formed during the process exclusively contains COX1, which acts as a seed for sequential incorporation of COX subunits. In this model, the addition of subunits COX4 and COX5a to S1 results in the progression to the second assembly intermediate (S2) (5,29,30). The accumulated subassembly in interfered cells corresponds with subcomplex S2 and indicates that hCOA3 is participating in the early steps of COX assembly, most probably facilitating the addition of the subsequent COX subunits to COX1 containing intermediates.…”