Key role of methionine-rich domains in copper homeostasis systems: focus on CueOs – MetCop

The MetCop project aims to investigate two fundamental questions related to the involvement of multicopper oxidases in copper homeostasis: (1) how methionine-rich (Met-rich) domains govern copper(I) oxidase activity and, consequently, copper detoxification; and (2) what the consequences of methionine oxidation within the Met-rich domains of CueO are for copper tolerance. The strength of this proposal lies in the combination of in vivo and in vitro approaches, as well as in the study of both synthetic models and a library of full-length proteins from different organisms. Results obtained from one approach are integrated with the others to enrich the overall understanding.

CueO enzymes from various microorganisms, exhibiting diverse structural features of their Met-rich domains and different levels of in vivo copper resistance, were selected. The copper-binding sites identified in the selected CueOs were reproduced in pseudopeptides. The copper-binding properties elucidated using these synthetic models guided CueO mutagenesis and experimental conditions to determine the copper-binding properties of CueO Met-rich domains. CueO enzymatic activity was then investigated. This approach made it possible to establish a clear correlation between in vitro CueO activity and in vivo copper resistance as a function of the properties of the Met-rich domains. The copper-binding affinity of methionine sulfoxide (Met-O) was also exploited to understand the role of Met-O in copper resistance in CueO enzymes.

One of the original features of the MetCop project is its multimodal and multiscale approach, ranging from in vitro molecular studies of the wild-type enzyme and mutants guided by copper-coordinating peptide models, to the in vivo evaluation of cellular copper resistance.

The respective affinities for Cu⁺ and Cu²⁺ of biomimetic compounds reproducing potential copper-binding sites within Met-rich domains were determined. These studies support the proposed mechanism for Cu⁺-oxidase activity, in which Cu⁺—and not Cu²⁺—binds to the methionine residues of the Met-rich domain. The low affinity of the peptides for Cu(I) further suggests that the Met-rich domain does not function as a true Cu(I) binding site, but rather as a “sponge” that transiently absorbs Cu(I) through a cooperative effect of the methionines within the domain.

Numerous mutants targeting copper-binding sites within the Met-rich domain were generated (Cu5, Cu6, and Cu7). Their activity was measured both in homogeneous solution using spectrophotometric assays with Cu⁺ complexes of varying affinities, and by electrochemistry after immobilization of the enzyme on an electrochemical interface.

Comparison of the solution kinetic constants of the wild-type protein and the mutants demonstrates that the domain contributes to Cu⁺-oxidase activity through a mechanism other than via the Cu6 and Cu7 sites. In contrast, deletion of two ligands at the Cu5 site results in an inactive enzyme, demonstrating the key role of Cu5 as the sole active site for Cu⁺ oxidation.

Electrochemical data confirm the central role of Cu5 in Cu⁺-oxidase activity and rule out Cu6 and Cu7 as active sites in CueO. However, mutants lacking the Met-rich domain are as active as the wild-type protein, in apparent contradiction with the homogeneous-phase data. A major difference between solution-based activity assays and electrochemical measurements is Cu⁺ complexation. It can therefore reasonably be proposed that the ensemble of methionine residues within the Met-rich domain—not only those directly coordinating Cu6 and Cu7—forms a transient complex that contributes to destabilizing strongly chelated Cu⁺ and facilitates its transfer to the Cu5 site.

To correlate in vitro activities with in vivo data, E. coli bacterial cells were complemented with the different mutants studied in vitro. Their resistance to increasing Cu²⁺ concentrations provides an indirect measure of CueO activity. These experiments demonstrate the key role played by Cu5 and the negligible effect of the Met-rich domain, suggesting weak Cu⁺ chelation.

Research conducted within the ANR MetCop project has highlighted the decisive role of methionine-rich (Met-rich) domains in the Cu⁺-oxidase activity of CueO enzymes from different origins. This original work was the subject of a science outreach communication on the national CNRS Chemistry website, thereby contributing to the visibility of the results among a broad audience. In addition, CueO was shown to contribute to copper resistance under anaerobic conditions, a mechanism that remains to be elucidated.

The MetCop project has also led to the initiation of two new research contracts. The first focuses on the development of an electrochemical biosensor for copper detection. The second is related to the flexibility of the Met-rich domain. Indeed, Met-rich domains are often composed of disordered loops. To date, only two computational studies have shown that the flexibility of CueO Met-rich domains impacts enzymatic activity. By definition, flexible domains lack a stable three-dimensional structure and are therefore challenging to analyze. Nevertheless, recent literature highlights the functional role of structural dynamics. AI-based methods such as AlphaFold are making progress in this area but require experimental validation. Advanced techniques (XFEL, NMR, or cryo-EM) provide conformational information but make it difficult to directly correlate these changes with enzymatic activity.

The project funded by the ANR in 2025 aims to develop a new concept to study protein domain motions in the presence of activators using electrochemical approaches.

Copper element is at the same time an essential micronutrient in living systems, being a cofactor of enzymes involved in many biological processes, but toxic for the cell when in excess. The understanding of mechanisms involved in intracellular copper balance between requirement and toxicity is thus crucial from fundamental and applicative points of view.
Among amino acids, methionine (Met) residues readily bind copper, and interestingly many proteins involved in copper homeostasis are rich in Met. As a model case, Escherichia coli CueO multi-copper oxidase, a periplasmic protein early recognised to be involved in copper resistance in vivo, presents a Met-rich domain proposed to be involved in the oxidation of Cu+ into the less toxic Cu2+. Intriguingly, such regions in CueOs greatly differ in term of structure and Met content according to the microorganisms.
The MetCop project aims at determining the role of CueO Met-rich domains in the mechanisms selected by various microorganisms along evolution to tackle copper stress. Two fundamental questions related to their involvement in copper homeostasis will be addressed: 1) To which extent Met-rich domains govern cuprous oxidase activity, hence copper detoxification? 2) What are the consequences of Met oxidation in Met-rich domains on copper tolerance? Through a multiscale and multidisciplinary approach, the MetCop project will allow to establish a clear correlation between i) structural features of Met-rich domains of a library of CueOs, ii) copper binding properties, iii) in vitro enzymatic activity and iv) in vivo copper resistance.
The multiscale approach set in the MetCop project involves entire cells, periplasmic extracts, purified proteins and synthetic peptide models, allowing a back and forth iterative process between cellular and molecular scales to answer the two fundamental targeted questions. More specifically, the molecular basis of Cu binding to pseudopeptides designed from identified Met-rich domains of CueOs, will improve our knowledge on copper-Met coordination and guide mutation of specific Met in the Met-rich domains of CueOs from various microorganisms. Changes in cuprous oxidase activities as a function of Met-rich domain structural features will be correlated to in vivo copper resistance. MetCop thus ambitions to precisely determine how and which Met(s) in Met-rich domains are involved in copper binding and copper resistance. Beyond the involvement in cuprous oxidase activity, MetCop will search for a role of Met(s) in Met-rich domains in the protection against reactive oxygen species that can be produced in the presence of copper. It will finally investigate whether MsrP, a protein recognized to reduce oxidized Met(s), may participate to maintain CueO activities in vivo.
The multidisciplinary approach is reflected by the panel of methodologies (some of them being especially set up in the MetCop project) carried out to determine the role of Met-rich domains of CueOs from different organisms in copper resistance: molecular biology, bioinformatics, biochemistry, chemical pseudopeptide design, theoretical methods and electrochemistry. This multidisciplinary approach is allowed thanks to a unique consortium with complementary expertise in i) genetics to study in vivo copper tolerance (LCB), ii) biophysics and biochemistry of redox enzymes including Cu-based metalloproteins (BIP), and iii) chemistry of copper binding to peptides (SYMMES).
Such a multiscale, multidisciplinary approach is expected to provide new findings that will pave the way to the understanding of the evolutive selection of Met residues at specific position within proteins involved in copper homeostasis.