Toward the mechanisms of copper insertion in plant Cytochrome c Oxidase"

MARÍA NATALIA LISA; PEDRO ALZARI; MARÍA EUGENIA LLASES; MARCOS N. MORGADA; ESTEFANÍA GIANNINI; ALEJANDRO J. VILA

Ventura

Conferencia; Gordon Research Conferences - Metals in Biology; 2019

Gordon Research Conferences

Copper is an essential cofactor of cytochrome c oxidase (COX), the terminal oxidase of the respiratory chain in most organisms. COX I and COX II are the two copper-containing subunits harboring the CuB and CuA sites, respectively, conserved in heme-copper oxidases. Assembly of the oxidase is a complex process involving the synthesis and folding of the individual subunits and the incorporation of the metal cofactors.1,2 Sco proteins belong to a family of proteins related to thioredoxins that contain two redox-active cysteine residues, and a histidine involved in copper binding.2 These proteins have been shown to be essential in the assembly of the CuA site, from subunit COX II. However, their role and the number of essential Sco orthologs differs among organisms. Indeed, the can act as Thiol-Oxidoreductases and/or copper chaperones in CuA assembly. Previously, we have proposed distinct mechanisms involving Sco proteins for the assembly of CuA sites in Bacterial and Human oxidases.3,4 Arabidopsis thaliana HHC1, is a protein from the Sco family that is essential for COX activity and plant viability.5,6 Here we report the biochemical, structural and functional characterization of this protein (renamed as AtSco1). AtSco1 is able to bind either Cu(I) or Cu(II) with high affinities (Kd10-18), in contrast to most Sco proteins. The crystal structure of the soluble fragment of Cu(I)-AtSco1 reveals a Cys2His coordination environment. The Cu(II) variant, instead, shows a tetrahedral environment, as revealed by several spectroscopic techniques. To test the biochemical function of AtSco1, we constructed a chimeric protein within the scaffold of the stable T. thermophilus COX II including the metal binding loops of the Arabidopsis COX II. Cu(II)-AtSco1 is able to elicit reduction of the oxidized oxidase, and transfer copper ions with a slow kinetics. Instead, Cu(I)-AtSco1 is an efficient copper metallochaperone for COX II, but is devoid of any redox activity either in the metallated or apo form. Overall, these data reveal that AtSco1 is a Cu(I) metallochaperone, while its role as oxidoreductase is still unclear. This picture provides a different scenario regarding Sco functions compared to that reported for the bacterial periplasmic proteins and the human mitochondrial ones. References1.Robinson, N. J. & Winge, D. R. Copper metallochaperones. Annu. Rev. Biochem. 79, 537?62 (2010).2.Leary, S. C. Redox regulation of SCO protein function: controlling copper at a mitochondrial crossroad. Antioxid. Redox Signal. 13, 1403?16 (2010).3.Morgada, M. N., Abriata, L. A., Cefaro, C., Gaijda, K. & Banci, L. Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase. 1?27 (2015).4.Abriata, L. A. et al. Nature Chemical Biology. 4, 599?601 (2009).5.Attallah, C. V et al. Plants contain two SCO proteins that are differentially involved in cytochrome c oxidase function and copper and redox homeostasis. J. Exp. Bot. 62, 4281?94 (2011).6.Steinebrunner, I., Gey, U., Andres, M., Garcia, L. & Gonzalez, D. H. Divergent functions of the Arabidopsis mitochondrial SCO proteins: HCC1 is essential for COX activity while HCC2 is involved in the UV-B stress response. Front. Plant Sci. 5, 87 (2014).