Measuring transverse relaxation in highly paramagnetic systems.

The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50-400 s-1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues.

Anion Recognition in Water, Including Sulfate, by a Bicyclam Bimetallic Receptor: A Process Governed by the Enthalpy/Entropy Compensatory Relationship.

The dimetallic system [CuII2 (L)]4+ contains two facing equivalent metallocyclam subunits and incorporates ambidentate anions, mono- (halides) and poly-atomic (sulfate), which bridge the two CuII centres. Isothermal titration calorimetry (ITC) experiments in water showed that the log K values of the inclusion equilibria for halides and sulfate varied over a restricted interval (3.6±0.2), which indicated lack of selectivity and that similarity of ΔG° values resulted from the unbalanced contribution of the ΔH° and TΔS° terms: the more favourable the one, the less favourable the other. In particular, a linear dependence of ΔH° and TΔS° was observed (a typical enthalpy/entropy compensatory diagram), which assigned a major role to hydration terms: 1) a more hydrated anion resulted in a more endothermic dehydration process; and 2) a larger number of water molecules released to the solution resulted in a more positive TΔS°. Limiting cases refer to the complexation 1) of the poorly hydrated iodide (highly exothermic process, entropically disfavoured), and 2) of the highly hydrated sulfate (moderately endothermic process, entropically very favoured). Anion receptors operating in water belong to two main domains: 1) those exhibiting positive ΔH° and positive TΔS° (+/+ signature), and 2) those displaying the opposite behaviour: (-/- signature). The receptor investigated herein connects the two domains, along the ΔH°/TΔS° straight line, thanks to the hidden role of the versatile metal-anion interaction.

Anion Binding by Dimetallic Nickel(II) and Nickel(III) Complexes of a Face-to-Face Bicyclam: Looking for a Bimacrocyclic Effect.

The dinickel(II) complex of the face-to-face bicyclam ligand α,α'-bis(5,7-dimethyl-1,4,8,11-tetraazacyclotetradecan-6-yl)-o-xylene (L∩L) in a dimethyl sulfoxide solution exists as a mixture of high- and low-spin forms and uptakes up to three halide and pseudohalide ions (X(-)), according to stepwise equilibria, whose constants were determined through spectrophotometric titration experiments. In the case of halides, the first anion goes into the intermetallic cavity, whereas pseudohalides first coordinate the metal center from outside. Comparison with equilibrium data for the complex with the macrocycle 5,7-dimethyl-6-benzyl-1,4,8,11-tetraazacyclotetradecane (L) shows that the dinuclear complex [Ni(II)2(L∩L)](4+) displays an affinity for the first halide distinctly higher than the mononuclear complex [Ni(II)(L)](2+), thus disclosing the existence of a bimacrocyclic effect for anion binding. Differential pulse voltammetry studies typically showed a three-peak profile: the most anodic pertaining to the [Ni(II)2(L∩L)](4+) → Ni(III)2(L∩L)](6+) two-electron process, then one originating from the [Ni(II)2(L∩L)X](3+) → Ni(III)2(L∩L)X](5+) two-electron process, and one deriving from the two two-electron half reactions [Ni(II)2(L∩L)X2](2+) → Ni(III)2(L∩L)X2](4+) and [Ni(II)2(L∩L)X3](+) → Ni(III)2(L∩L)X3](3+), taking place at nearly the same potential. The crystal structure of the [Ni(II)2(L∩L)(µ-NCO)(NCO)2]ClO4·2.5H2O complex salt showed a caterpillar arrangement of the three metal-bound cyanate ions.

Interconversion between [2Fe-2S] and [4Fe-4S] cluster glutathione complexes.

We present here how different iron-sulfide-glutathione ratios, applied in in vitro conditions comparable to those present in the mitochondrial matrix, affect the speciation of iron-sulfur cluster glutathione complexes. An excess of sulfide with respect to iron ions promotes the formation of a tetranuclear [FeII2FeIII2S4(GS)4]2- complex, while an excess of iron ions favors the formation of a dinuclear [FeIIFeIIIS2(GS)4]3- complex. These two complexes establish an interconversion equilibrium. The latter might play a role in the composition of the mitochondrial labile iron pool potentially contributing to the regulation of cellular iron homeostasis.

PRE-driven protein NMR structures: an alternative approach in highly paramagnetic systems.

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

1H, 13C and 15N assignment of the paramagnetic high potential iron-sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1.

High potential iron-sulfur proteins (HiPIPs) are a class of small proteins (50-100 aa residues), containing a 4Fe-4S iron-sulfur cluster. The 4Fe-4S cluster shuttles between the oxidation states [Fe4S4]3+/2+, with a positive redox potential in the range (500-50 mV) throughout the different known HiPIPs. Both oxidation states are paramagnetic at room temperature. HiPIPs are electron transfer proteins, isolated from photosynthetic bacteria and usually provide electrons to the photosynthetic reaction-center. PioC, the HIPIP isolated from Rhodopseudomonas palustris TIE-1, is the smallest among all known HiPIPs. Despite their small dimensions, an extensive NMR assignment is only available for two of them, because paramagnetism prevents the straightforward assignment of all resonances. We report here the complete NMR assignment of 1H, 13C and 15N signals for the reduced [Fe4S4]2+ state of the protein. A set of double and triple resonance experiments performed with standardized parameters/datasets provided the assignment of about 72% of the residues. The almost complete resonance assignment (99.5% of backbone and ca. 90% of side chain resonances) was achieved by combining the above information with those obtained using a second set of NMR experiments, in which acquisition and processing parameters, as well as pulse sequences design, were optimized to account for the peculiar features of this paramagnetic protein.

Bimacrocyclic Effect in Anion Recognition by a Copper(II) Bicyclam Complex.

The dicopper(II) complex of the bimacrocyclic ligand α,α'-bis(5,7-dimethyl-1,4,8,11-tetraazacyclotetradecan-6-yl)-o-xylene, 2, interacts with selected anions in dimethyl sulfoxide solution according to two different modes: (i) halides (Cl-, Br-, and I-) and N3 - coordinate the two metal centers at the same time between the two macrocyclic subunits that face each other and (ii) anionic species that do not fit the bridging coordination mode (e.g., NCO-, SCN-, CH3COO-, NO3 -, and H2PO4 -) interact with copper(II) ions only at the "external" positions or their interaction is too weak to be detected. Occurrence of the bridging interaction is demonstrated by X-ray crystallographic studies performed on the adduct formed by [Cu2(2)]4+ with azide and by electron paramagnetic resonance investigation, as the anion coordination between the two copper(II) centers induces spin-spin coupling. Isothermal titration calorimetry experiments performed on [Cu2(2)]4+ and, for comparison, on [(5,7-dimethyl-6-benzyl-1,4,8,11-tetraazacyclotetradecane)copper(II)], representing the mononuclear analogue, allowed determination of thermodynamic parameters (log K, ΔH, and TΔS) associated with the considered complex/anion equilibria. Thermodynamic data showed that adducts formed by [Cu2(2)]4+ with halides and azide benefit from an extra stability that can be explained on the basis of the anion advantage of simultaneously binding the two metal centers, i.e., in terms of the bimacrocyclic effect.