Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase C. Oswald, U. Krause-Buchholz⁎ and G. Rödel Institute of Genetics, Dresden University of Technology, 01062 Dresden, Germany Received 20 January 2009; received in revised form 9 April 2009; accepted 16 April 2009 Available online 22 April 2009 Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel elec- trophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼ 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated syn- thesis and association of individual complexes, is proposed. © 2009 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: cytochrome c oxidase; Cox17; mitochondria; supercomplex assembly; siRNA Introduction The oxidative phosphorylation system (OXPHOS) in mitochondria (mt) provides a highly efficient route to generate ATP from energy-rich molecules. Electrons are transported in the respiratory chain (RC) from NADH or succinate to complex I (NADH ubiquinone oxidoreductase) or complex II (succi- nate ubiquinone oxidoreductase), respectively, and further via complex III (ubiquinol cytochrome c oxidoreductase) and complex IV (cytochrome c oxidase, COX) to the terminal acceptor molecular oxygen. 1 Complexes I, III and IV couple electron transport with the generation of a proton gradient across the inner mt membrane. 2 Complex V (F 1 F 0 - ATP synthase) utilizes the proton gradient to syn- thesize ATP. Eukaryotic COX is a multisubunit enzyme composed of 11 or more subunits, depending on the organism. 3 Subunits Cox1, Cox2 and Cox3 are encoded by the mt genome and form the catalytic core of COX. Insertion of essential cofactors, among them copper for the two redox-active metal centres Cu A and Cu B in subunit Cox1 and Cox2, respectively, as well as the coordinated assembly of imported nuclear encoded and of mt synthesized subunits is essential for the formation of a functional enzyme complex. In the absence of the copper centres, subunits Cox1 and Cox2 are degraded rapidly and the COX holoenzyme fails to assemble. 4,5 A number of copper-binding pro- teins, like Cox17, Sco1 and Sco2 are required for the uptake, transfer and delivery of copper ions to the COX subunits. The copper chaperone Cox17 was initially identified and characterized in the yeast Saccharomyces cerevisiae. 6 Rescue of the cox17 mutation by over-expression of either SCO1 or SCO2 led to the conclusion that these two proteins act downstream of Cox17 in the mt copper metabolism. 7 *Corresponding author. E-mail address: Udo.Krause-Buchholz@tu-dresden.de. Abbreviations used: OXPHOS, oxidative phosphorylation system; mt, mitochondria; RC, respiratory chain; COX, cytochrome c oxidase; IMS, intermembrane space; BN, blue native; SC, supercomplex; CS, citrate synthase; RT, room temperature; PIC, protease inhibitor cocktail; DAB, diaminobenzidine; TCEP, tris(2- carboxyethyl)phosphine. doi:10.1016/j.jmb.2009.04.034 J. Mol. Biol. (2009) 389, 470–479 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.