International audienceno abstrac

Selective 15N isotope labeling of the cytochrome bo3 ubiquinol oxidase from E. coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from R71, H98, and Q101 residues and peptide nitrogens from R71 and H98 residues around the semiquinone (SQ) at the high-affinity QH site. The 2D ESEEM (HYSCORE) data have directly identified the Nε of R71 as an H-bond donor carrying the largest amount of the unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the QH site and strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that the Nε from a histidine residue, presumably H75, carries most of the unpaired spin density instead of the Nε of R71, as in the wild-type bo3. However, the detailed characterization of the weakly coupled 15Ns from selective labeling of R71 and Q101 in D75H was precluded by overlap of the 15N lines with the much stronger ~1.6 MHz line from quadrupole triplet of the strongly coupled 14Nε from H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied in order to probe the contribution of R71 and Q101 to the 15N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory based calculations to model the available information about 1H, 15N and 13C hyperfine couplings for the QH site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone’s electron transfer function.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)(Cys) (n = 0, 1, 2) cluster by selective cysteine-C isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe ligands than the nonreducible Fe ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-C hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

Michael addition (MA) is one of the most well studied chemical transformation in synthetic chemistry. Here, we report the synthesis and crystal structures of a library of 3d/4f coordination clusters (CCs) formulated as [ZnYL(solv)(Z)] and study their catalytic properties toward the MA of nitrostyrenes with barbituric acid derivatives. Each CC presents two borderline hard/soft Lewis acidic Zn centers and two hard Lewis acidic Y centers in a defect dicubane topology that brings the two different metals into a proximity of ∼3.3 Å. Density functional theory computational studies suggest that these tetrametallic CCs dissociate in solution to give two catalytically active dimers, each containing one 3d and one 4f metal that act cooperatively. The mechanism of catalysis has been corroborated via NMR, electron paramagnetic resonance, and UV-vis. The present work demonstrates for the first time the successful use of 3d/4f CCs as efficient and high diastereoselective catalysts in MA reactions.