Search

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.

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.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CueP confers bacterial copper resistance in the periplasm, particularly under anaerobic conditions, through an unknown mechanism. The only available structure and limited solution data suggest that CueP forms noncovalent dimers in solution, whereas sequence conservation suggests important roles for three cysteines and two histidines as copper ligands. Here we report evidence of a dimerization equilibrium mediated by a newly identified interface of functional relevance, which occludes internal copper sites and disulfide bonds but allows for intra- and interchain disulfide bonding, an extensive disulfide relay, and interfacial copper sites. Our results suggest a role for CueP linking redox-state sensing and copper detoxification., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cupredoxins perform copper-mediated long-range electron transfer (ET) in biological systems. Their copper-binding sites have evolved to force copper ions into ET-competent systems with decreased reorganization energy, increased reduction potential, and a distinct electronic structure compared with those of non-ET-competent copper complexes. The entatic or rack-induced state hypothesis explains these special properties in terms of the strain that the protein matrix exerts on the metal ions. This idea is supported by X-ray structures of apocupredoxins displaying "closed" arrangements of the copper ligands like those observed in the holoproteins; however, it implies completely buried copper-binding atoms, conflicting with the notion that they must be exposed for copper loading. On the other hand, a recent work based on NMR showed that the copper-binding regions of apocupredoxins are flexible in solution. We have explored five cupredoxins in their "closed" apo forms through molecular dynamics simulations. We observed that prearranged ligand conformations are not stable as the X-ray data suggest, although they do form part of the dynamic landscape of the apoproteins. This translates into variable flexibility of the copper-binding regions within a rigid fold, accompanied by fluctuations of the hydrogen bonds around the copper ligands. Major conformations with solvent-exposed copper-binding atoms could allow initial binding of the copper ions. An eventual subsequent incursion to the closed state would result in binding of the remaining ligands, trapping the closed conformation thanks to the additional binding energy and the fastening of noncovalent interactions that make up the rack.

Previous studies on engineered CuA centres have shown that one of the histidine ligands is protonated and dissociated from the metal site at physiological pH values, thus suggesting a role in regulating proton-coupled electron transfer of cytochrome c oxidases in vivo. Here we report that for native CuA such protonation does not take place at physiologically relevant pH values and, furthermore, no significant changes in the spectroscopic and redox properties of the metal site occur at low pH.

An updated picture of the ligand sets and copper-ligand atom bond lengths in proteins is presented which takes advantage of (i) the approximately twofold increase in the number of entries for copper-containing proteins in the PDB since the last study of this kind, especially benefiting from the recent incorporation of the structures of proteins involved in copper homeostasis, and (ii) a preliminary classification of copper sites based on their structural, electronic and functional features. This classification allowed the calculation of reliable target copper-ligand distances for several bonds that were not available in previous work and that are in good agreement with EXAFS data and the known chemistry of these sites. The analysis presented here further disclosed an artifactual dependence of the average of the reported Cu-NHis bond lengths on structure resolution, highlighting the importance of taking this into account when computing target distances even from high-resolution structures. Finally, a relationship between the two Cu-O distances in bidentate carboxylates is disclosed, similar to that reported previously for other metal ions.

The electronic properties of Thermus thermophilus Cu(A) in the oxidized form were studied by (1)H and (13)C NMR spectroscopy. All of the (1)H and (13)C resonances from cysteine and imidazole ligands were observed and assigned in a sequence-specific fashion. The detection of net electron spin density on a peptide moiety is attributed to the presence of a H-bond to a coordinating sulfur atom. This hydrogen bond is conserved in all natural Cu(A) variants and plays an important role for maintaining the electronic structure of the metal site, rendering the two Cys ligands nonequivalent. The anomalous temperature dependence of the chemical shifts is explained by the presence of a low-lying excited state located about 600 cm(-1) above the ground state. The room-temperature shifts can be described as the thermal average of a sigma(u)* ground state and a pi(u) excited state. These results provide a detailed description of the electronic structure of the Cu(A) site at atomic resolution in solution at physiologically relevant temperature.

Copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. Here we show that a new periplasmic protein (PCu(A)C) selectively inserts Cu(I) ions into subunit II of Thermus thermophilus ba(3) oxidase to generate a native Cu(A) site. The purported metallochaperone Sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the Cu(A) cysteine ligands.