The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro.

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu+ and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu+-transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.

CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential.

Attaining rational modulation of thermodynamic and kinetic redox parameters of metalloproteins is a key milestone towards the (re)design of proteins with new or improved redox functions. Here we report that implantation of ligand loops from natural T1 proteins into the scaffold of a CuA protein leads to a series of distorted T1-like sites that allow for independent modulation of reduction potentials (E°') and electron transfer reorganization energies (λ). On the one hand E°' values could be fine-tuned over 120 mV without affecting λ. On the other, λ values could be modulated by more than a factor of two while affecting E°' only by a few millivolts. These results are in sharp contrast to previous studies that used T1 cupredoxin folds, thus highlighting the importance of the protein scaffold in determining such parameters.

Purple Mixed-Valent Copper A.

CuA is a binuclear copper center acting as an electron transfer hub in terminal oxidases such as cytochrome c oxidase and nitrous oxide reductase. Its unique electronic structure is intimately linked to its function and has puzzled the community of biological inorganic chemistry for decades. Here we review the insights provided by different spectroscopic techniques of CuA centers, and the different experimental approaches to tackle its study, that encompass the synthesis of model compounds as well as protein engineering efforts. The contribution of the electronic structure to the thermodynamic and kinetic of electron transfer is extensively discussed. We also describe the proposed mechanism of CuAassembly in different organisms. The recent discovery of a novel CuA site opens new perspectives to this field.

Unexpected electron spin density on the axial methionine ligand in CuA suggests its involvement in electron pathways.

The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways.

Arabidopsis thaliana Hcc1 is a Sco-like metallochaperone for CuA assembly in Cytochrome c Oxidase.

The assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1 -bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxn H motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities. DATABASE: PDB entry 6N5U (Crystal structure of Arabidopsis thaliana ScoI with copper bound).

COA6 Is Structurally Tuned to Function as a Thiol-Disulfide Oxidoreductase in Copper Delivery to Mitochondrial Cytochrome c Oxidase.

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coil-helix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.

pH-Induced Binding of the Axial Ligand in an Engineered CuA Site Favors the πu State.

CuA centers perform efficient long-range electron transfer. The electronic structure of native CuA sites can be described by a double-potential well with a dominant σu* ground state in fast equilibrium with a less populated πu ground state. Here, we report a CuA mutant in which a lysine was introduced in the axial position. This results in a highly unstable protein with a pH-dependent population of the two ground states. Deep analysis of the high-pH form of this variant shows the stabilization of the πu ground state due to direct binding of the Lys residue to the copper center that we attribute to deprotonation of this residue.

Biochemistry of Copper Site Assembly in Heme-Copper Oxidases: A Theme with Variations.

Copper is an essential cofactor for aerobic respiration, since it is required as a redox cofactor in Cytochrome c Oxidase (COX). This ancient and highly conserved enzymatic complex from the family of heme-copper oxidase possesses two copper sites: CuA and CuB. Biosynthesis of the oxidase is a complex, stepwise process that requires a high number of assembly factors. In this review, we summarize the state-of-the-art in the assembly of COX, with special emphasis in the assembly of copper sites. Assembly of the CuA site is better understood, being at the same time highly variable among organisms. We also discuss the current challenges that prevent the full comprehension of the mechanisms of assembly and the pending issues in the field.

Formation and Electronic Structure of an Atypical CuA Site.

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper CuA center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD CuA bear similarities to those of canonical CuA sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric CuA site to two mononuclear Cu2+ sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD CuA formation occurs rapidly and involves a short-lived intermediate with a λmax of 360 nm. Unlike other CuA sites, the PmoD CuA is unstable, decaying to two type 2 Cu2+ centers. Surprisingly, NMR data indicate that the PmoD CuA has a pure σu* ground state rather than the typical equilibrium between σu* and πu of all other CuA proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the CuA core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known CuA sites.

Fine Tuning of Functional Features of the CuA Site by Loop-Directed Mutagenesis.

Here we report the spectroscopic and electrochemical characterization of three novel chimeric CuA proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between CuA sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of CuA.. This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter.

Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes.

CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.

Tuning of Enthalpic/Entropic Parameters of a Protein Redox Center through Manipulation of the Electronic Partition Function.

Manipulation of the partition function (Q) of the redox center CuA from cytochrome c oxidase is attained by tuning the accessibility of a low lying alternative electronic ground state and by perturbation of the electrostatic potential through point mutations, loop engineering and pH variation. We report clear correlations of the entropic and enthalpic contributions to redox potentials with Q and with the identity and hydrophobicity of the weak axial ligand, respectively.

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

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.

Reversible Switching of Redox-Active Molecular Orbitals and Electron Transfer Pathways in Cu(A) Sites of Cytochrome†c Oxidase.

The Cu(A) site of cytochrome c oxidase is a redox hub that participates in rapid electron transfer at low driving forces with two redox cofactors in nearly perpendicular orientations. Spectroscopic and electrochemical characterizations performed on first and second-sphere mutants have allowed us to experimentally detect the reversible switching between two alternative electronic states that confer different directionalities to the redox reaction. Specifically, the M160H variant of a native Cu(A) shows a reversible pH transition that allows to functionally probe both states in the same protein species. Alternation between states exerts a dramatic impact on the kinetic redox parameters, thereby suggesting this effect as the mechanism underlying the efficiency and directionality of Cu(A) electron transfer in vivo. These findings may also prove useful for the development of molecular electronics.

Control of the electronic ground state on an electron-transfer copper site by second-sphere perturbations.

The Cu(A) center is a dinuclear copper site that serves as an optimized hub for long-range electron transfer in heme-copper terminal oxidases. Its electronic structure can be described in terms of a σ(u)* ground-state wavefunction with an alternative, less populated ground state of π(u) symmetry, which is thermally accessible. It is now shown that second-sphere mutations in the Cu(A) containing subunit of Thermus thermophilus ba3 oxidase perturb the electronic structure, which leads to a substantial increase in the population of the π(u) state, as shown by different spectroscopic methods. This perturbation does not affect the redox potential of the metal site, and despite an increase in the reorganization energy, it is not detrimental to the electron-transfer kinetics. The mutations were achieved by replacing the loops that are involved in protein-protein interactions with cytochrome c, suggesting that transient protein binding could also elicit ground-state switching in the oxidase, which enables alternative electron-transfer pathways.