Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features.

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.

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.

New trends in the chemistry of iron(III) citrate complexes: correlations between X-ray structures and solution species probed by electrospray mass spectrometry and kinetics of iron uptake from citrate by iron chelators.

Despite the crucial role of "iron(III) citrate systems" in the iron metabolism of living organisms (bacteria as well as plants or mammals), the coordination chemistry of ferric citrate remains poorly defined. Variations in the experimental conditions used for the preparation of so-called ferric citrates (iron salt, Fe:cit molar ratio, base, pH, temperature, solvent) lead to several different species, which are in equilibrium in solution. To date, six different anionic complexes have been structurally characterized in the solid state, by ourselves or others. In the work described herein, we have established the experimental conditions leading to each of them. Five were obtained from aqueous solution. With the exception of a nonanuclear species (of which fragments have been detected), all were identified in aqueous solution on the basis of electrospray ionization mass spectrometry. In addition, the spectra revealed a new trinuclear species, which could not be crystallized. Kinetic studies of iron uptake from citrate species by iron chelators confirmed the results indicated by the ESI-MS studies. These studies also allowed the relative molar fraction of mononuclear versus polynuclear complexes to be determined, which depends on the Fe:cit molar ratio.

Tetrathiafulvalene-phosphine-based iron and ruthenium carbonyl complexes: electrochemical and EPR studies.

The radical cation of the redox active ligand 3,4-dimethyl-3',4'-bis-(diphenylphosphino)-tetrathiafulvalene (P2) has been chemically and electrochemically generated and studied by EPR spectroscopy. Consistent with DFT calculations, the observed hyperfine structure (septet due to the two methyl groups) indicates a strong delocalization of the unpaired electron on the central S2C=CS2 part of the tetrathiafulvalene (TTF) moiety and zero spin densities on the phosphine groups. In contrast with the ruthenium(0) carbonyl complexes of P2 whose one-electron oxidation directly leads to decomplexation and produces P2*+, one-electron oxidation of [Fe(P2)(CO)3] gives rise to the metal-centered oxidation species [Fe(I)(P2)(CO)3], characterized by a coupling with two 31P nuclei and a rather large g-anisotropy. The stability of this complex is however modest and, after some minutes, the species resulting from the scission of a P-Fe bond is detected. Moreover, in presence of free ligand, [Fe(I)(P2)(CO)3] reacts to give the complex [Fe(I)(P2)2(CO)] containing two TTF fragments. The two-electron oxidation of [Fe(P2)(CO)3] leads to decomplexation and to the P2*+ spectrum. Besides EPR spectroscopy, cyclic voltammetry as well as FTIR spectroelectrochemistry are used in order to explain the behaviour of [Fe(P2)(CO)3] upon oxidation. This behaviour notably differs from that of the Ru(0) counterpart. This difference is tentatively rationalized on the basis of structural arguments.

Comparative studies on the iron chelators O-TRENSOX and TRENCAMS: selectivity of the complexation towards other biologically relevant metal ions and Al(3+).

Complexation constants have been determined by potentiometric titration and spectrophotometric measurements for several biologically relevant divalent metals (Ca(2+), Cu(2+), Zn(2+)) as well as Al(3+) with the sulfonated tris(8-hydroxyquinolinate) tripodal ligand O-TRENSOX. The values demonstrate great selectivity of O-TRENSOX for Fe(3+) according to the sequence Fe(3+) >>Cu(2+)>Zn(2+)>Ca(2+). This selectivity is compared to that shown by tris(hydroxamate) and tris(catecholate) ligands. (1)H NMR spectroscopy of the diamagnetic complexes have been carried out in (2)H(2)O solutions.