Site-directed mutagenesis reveals a conservation of the copper-binding site and the crucial role of His24 in CopH from Cupriavidus metallidurans CH34.

CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study.

Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent.

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.

CopH from Cupriavidus metallidurans CH34. A novel periplasmic copper-binding protein.

The copH gene is one of the 19 open reading frames (ORFs) found in the cop cluster borne by the large plasmid pMol30 in Cupriavidus metallidurans CH34. The entire cluster is involved in detoxification of copper from the cytoplasm as well as from the periplasm. The function of the corresponding protein, CopH, is not yet clear, but it seems to be involved in the late response phase. We have cloned copH and overproduced and purified the corresponding protein. CopH is rather unique as only one paralog can be found in the databases. It is a dimeric protein with a molecular mass of 13 200 Da per subunit and located in the periplasm. The metal binding properties of CopH were examined by using a series of techniques such as UV-visible spectroscopy, circular dichroism (CD), electron paramagnetic resonance (EPR), surface plasmon resonance (SPR), mass spectrometry, and nuclear magnetic resonance (NMR). All together, the corresponding data are consistent with a dimeric protein containing one metal-binding site per subunit. These sites have a high affinity for Cu(II) but can also bind zinc or nickel. CopH does not contain any cysteines or methionines but contains two histidines. EPR and UV-visible features are consistent with the presence of Cu(II) type 2 centers in a nitrogen ligand field. SPR data confirm the involvement of the histidine residues in copper binding. CD and NMR data reveal that CopH is partially unfolded.