A comparative structural genomic analysis of a new class of metal-trafficking proteins can provide insights into the intracellular chemistry of reactive cofactors such as copper and zinc. Starting from the sequences of the metallochaperone Atx1 and from the first soluble domain of the copper-transporting ATPase Ccc2, both from yeast, a search on the available genomes was performed using a homology criterion and a metal-binding motif x‘-x"-C-x‴-x⁗-C. By limiting ourselves to 20% identity with any of the proteins found, several soluble copper-transport proteins were identified, as well as soluble domains of membrane-bound ATPases. Structural models were calculated using high-resolution solution structures as templates, and the models were validated using statistical and energy criteria. Residue conservation and substitution have been interpreted and discussed in terms of structure–function relationship. The potential energy surfaces have been analyzed in terms of protein–protein interactions. We find that metallochaperones and their physiological partner ATPases from several phylogenetic kingdoms recognize one another, via an interplay of electrostatics, hydrogen bonding, and hydrophobic interactions, in a manner that precisely orients the metal-binding side chains for rapid metal transfer between otherwise tight binding sites. Finally, other putative metal-transport proteins are mentioned that have low homology and/or a different metal-binding consensus motif and that appear to use similar structures for recognition and transfer. This analysis highlights the wealth and the complexity of the field.

The human protein Cox17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S–S bonds and two reduced cysteines (HCox17 _2S-S ). HCox17 _2S-S is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCox17 _2S-S , i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCox17 _3S-S , which can be then reduced by glutathione to apoHCox17 _2S-S . The HSco1/HCox17 _2S-S redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper–electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein–protein complex, which is instead observed in the Cu(I)HCox17 _2S-S /HSco1 interaction.

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.