Nickel Meets Aryl Thianthrenium Salts: Ni(I)-Catalyzed Halogenation of Arenes.

Herein, a regioselective, late-stage two-step arene halogenation method is reported. We propose how unusual Ni(I)/(III) catalysis is enabled by a combination of aryl thianthrenium and Ni redox properties that is hitherto unachieved with other (pseudo)halides. The catalyst is accessed in situ from inexpensive NiCl2·6(H2O) and zinc without the need of supporting ligands.

Dialkyl Ether Formation at High-Valent Nickel.

In this article, we investigated the I2-promoted cyclic dialkyl ether formation from 6-membered oxanickelacycles originally reported by Hillhouse. A detailed mechanistic investigation based on spectroscopic and crystallographic analysis revealed that a putative reductive elimination to forge C(sp3)-OC(sp3) using I2 might not be operative. We isolated a paramagnetic bimetallic NiIII intermediate featuring a unique Ni2(OR)2 (OR = alkoxide) diamond-like core complemented by a µ-iodo bridge between the two Ni centers, which remains stable at low temperatures, thus permitting its characterization by NMR, EPR, X-ray, and HRMS. At higher temperatures (>-10 °C), such bimetallic intermediate thermally decomposes to afford large amounts of elimination products together with iodoalkanols. Observation of the latter suggests that a C(sp3)-I bond reductive elimination occurs preferentially to any other challenging C-O bond reductive elimination. Formation of cyclized THF rings is then believed to occur through cyclization of an alcohol/alkoxide to the recently forged C(sp3)-I bond. The results of this article indicate that the use of F+ oxidants permits the challenging C(sp3)-OC(sp3) bond formation at a high-valent nickel center to proceed in good yields while minimizing deleterious elimination reactions. Preliminary investigations suggest the involvement of a high-valent bimetallic NiIII intermediate which rapidly extrudes the C-O bond product at remarkably low temperatures. The new set of conditions permitted the elusive synthesis of diethyl ether through reductive elimination, a remarkable feature currently beyond the scope of Ni.

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe-2S] Proteins.

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxin-like ferredoxin (TLF) domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi, and unicellular pathogenic eukaryotes but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability of yeast cells upon treatment with the redox mediators gallobenzophenone or pyrogallol, respectively. UV-vis, EPR, and Mössbauer spectroscopy of purified wild-type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion, and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 ( Em,7 = -164 ± 5 mV, p Kox1,2 = 7.9 ± 0.1 and 9.7 ± 0.1) differs from both dioxygenase ( Em,7 ≈ -150 mV, p Kox1,2 = 9.8 and 11.5) and cytochrome bc1/ b6 f Rieske clusters ( Em,7 ≈ +300 mV, p Kox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands, or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of the [2Fe-2S] cluster containing proteins with bishistidinyl coordination.

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy.

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN- and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.

Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases.

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN- ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S]H2+ ⇔ [4Fe-4S]H+ and [Fe(I)Fe(II)]H ⇔ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H+ + 2e- ⇄ H2. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]H+ - [Fe(I)Fe(II)]H and HredH+ = [4Fe-4S]H2+ - [Fe(I)Fe(I)]H (H+) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (Hred/HredH+) has a pKa ≈ 7.2.

Importance of Hydrogen Bonding in Fine Tuning the [2Fe-2S] Cluster Redox Potential of HydC from Thermotoga maritima.

Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the TmHydC subunit of the trimeric [FeFe]-hydrogenase from Thermotoga maritima, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of -526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron-sulfur cluster protein folds at binding different types of clusters.

Inactivation of the conserved open reading frame ycf34 of Synechocystis sp. PCC 6803 interferes with the photosynthetic electron transport chain.

Ycf34 is a hypothetical chloroplast open reading frame that is present in the chloroplast genomes of several non-green algae. Ycf34 homologues are also encoded in all sequenced genomes of cyanobacteria. To evaluate the role of Ycf34 we have constructed and analysed a cyanobacterial mutant strain. Inactivation of ycf34 in Synechocystis sp. PCC 6803 showed no obvious phenotype under normal light intensity growth conditions. However, when the cells were grown under low light intensity they contained less and smaller phycobilisome antennae and showed a strongly retarded growth, suggesting an essential role of the Ycf34 polypeptide under light limiting conditions. Northern blot analysis revealed a very weak expression of the phycocyanin operon in the ycf34 mutant under light limiting growth in contrast to the wild type and to normal light conditions. Oxygen evolution and P(700) measurements showed impaired electron flow between photosystem II and photosystem I under these conditions which suggest that the impaired antenna size is most likely due to a highly reduced plastoquinone pool which triggers regulation on a transcriptional level. Using a FLAG-tagged Ycf34 we found that this protein is tightly bound to the thylakoid membranes. UV-vis and Mössbauer spectroscopy of the recombinant Ycf34 protein demonstrate the presence of an iron-sulphur cluster. Since Ycf34 lacks homology to known iron-sulphur cluster containing proteins, it might constitute a new type of iron-sulphur protein implicated in redox signalling or in optimising the photosynthetic electron transport chain.

Radical mechanism of cyanophage phycoerythrobilin synthase (PebS).

PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.

The unusal redox centers of SoxXA, a novel c-type heme-enzyme essential for chemotrophic sulfur-oxidation of Paracoccus pantotrophus.

The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.