A periplasmic cupredoxin with a green CuT1.5 center is involved in bacterial copper tolerance.

The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu-resistant cuproprotein CopI is present in many human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine, and methionine residues that could be ligands for different copper binding sites, among which the cupredoxin center could be involved in the protein function. Here, we demonstrated that Vibrio cholerae and Pseudomonas aeruginosa CopI restore the Cu-resistant phenotype in the Rubrivivax gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, and (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single-domain cupredoxin with more than one Cu site involved in Cu resistance.

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α-Helical Metalloprotein.

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.

Impact of copper ligand mutations on a cupredoxin with a green copper center.

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

The hyperthermophilic anaerobe Thermotoga Maritima is able to cope with limited amount of oxygen: insights into its defence strategies.

Thermotoga maritima, an anaerobic hyperthermophilic bacterium, was found able to grow in the presence of low concentrations of oxygen of up to 0.5% (v/v). Differential proteomics and transcripts analysis by qRT-PCR were used to identify the defence strategies used by T. maritima to protect itself against oxygen. A flavoprotein, homologous to rubredoxin oxygen oxidoreductase was found to be overproduced when cells were cultured in oxidative conditions. The recombinant protein, produced in Escherichia coli, exhibited an oxygen reductase activity, which could account for the observed decrease in oxygen concentration during growth. The gene encoding this oxygen reductase belongs to a multicistronic unit that includes genes encoding proteins involved in exopolysaccharide biosynthesis, which may be related to a biofilm formation induced by the presence of oxygen. Enzymes involved in reactive oxygen species detoxification, iron-sulfur centre synthesis/repair and the cysteine biosynthesis pathway were also overproduced. All these enzymatic systems together contribute to the defence strategy of T. maritima against oxygen. Because of the position of T. maritima in deep branches of the phylogenetic tree, we suggest that these strategies can be considered as ancestral mechanisms first developed by anaerobic microorganisms on the early Earth to protect themselves against primary abiotic or biotic oxygen production.