Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of CuA in human cytochrome oxidase

Marcos N Morgada,Luciano A Abriata,Chiara Cefaro,Karolina Gajda,Lucia Banci,Alejandro J Vila

doi:10.1073/pnas.1505056112

Abstract

Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors. Two orthologous Sco proteins (Sco1 and Sco2) are essential for the correct assembly of the dicopper CuA site in the human oxidase, but their function is not fully understood. Here, we report an in vitro biochemical study that shows that Sco1 is a metallochaperone that selectively transfers Cu(I) ions based on loop recognition, whereas Sco2 is a copper-dependent thiol reductase of the cysteine ligands in the oxidase. Copper binding to Sco2 is essential to elicit its redox function and as a guardian of the reduced state of its own cysteine residues in the oxidizing environment of the mitochondrial intermembrane space (IMS). These results provide a detailed molecular mechanism for CuA assembly, suggesting that copper and redox homeostasis are intimately linked in the mitochondrion.

Highlights

Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors
We posit a mechanism of assembly of the CuA site in human c oxidase (COX) II that allows us to propose specific, complementary roles for Sco1 and Sco2
We show that (i) Sco1 is the metallochaperone that delivers the two Cu(I) equivalents to COX II; (ii) Sco1 and COX II have evolved specific protein– protein recognition based on the sequences of the loops containing the copper ligands; and (iii) Sco2 is responsible of reducing the Cys residues of COX II, but it performs this redox function in its Cu (I)-bound form rather than through a simple disulfide exchange reaction as observed in bacteria

Summary

Introduction

Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors. We show that (i) Sco is the metallochaperone that delivers the two Cu(I) equivalents to COX II; (ii) Sco and COX II have evolved specific protein– protein recognition based on the sequences of the loops containing the copper ligands; and (iii) Sco is responsible of reducing the Cys residues of COX II, but it performs this redox function in its Cu (I)-bound form rather than through a simple disulfide exchange reaction as observed in bacteria This model reveals the versatility of the Sco scaffold in fulfilling different copper-dependent functions and, at the same time, providing high selectivity in the metal homeostasis network

Methods

Results

Conclusion