Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

The ABCB7-Like Transporter PexA in Rhodobacter capsulatus Is Involved in the Translocation of Reactive Sulfur Species.

The mitochondrial ATP-binding cassette (ABC) transporters ABCB7 in humans, Atm1 in yeast and ATM3 in plants, are highly conserved in their overall architecture and particularly in their glutathione binding pocket located within the transmembrane spanning domains. These transporters have attracted interest in the last two decades based on their proposed role in connecting the mitochondrial iron-sulfur (Fe-S) cluster assembly with its cytosolic Fe-S cluster assembly (CIA) counterpart. So far, the specific compound that is transported across the membrane remains unknown. In this report we characterized the ABCB7-like transporter Rcc02305 in Rhodobacter capsulatus, which shares 47% amino acid sequence identity with its mitochondrial counterpart. The constructed interposon mutant strain in R. capsulatus displayed increased levels of intracellular reactive oxygen species without a simultaneous accumulation of the cellular iron levels. The inhibition of endogenous glutathione biosynthesis resulted in an increase of total glutathione levels in the mutant strain. Bioinformatic analysis of the amino acid sequence motifs revealed a potential aminotransferase class-V pyridoxal-5'-phosphate (PLP) binding site that overlaps with the Walker A motif within the nucleotide binding domains of the transporter. PLP is a well characterized cofactor of L-cysteine desulfurases like IscS and NFS1 which has a role in the formation of a protein-bound persulfide group within these proteins. We therefore suggest renaming the ABCB7-like transporter Rcc02305 in R. capsulatus to PexA for PLP binding exporter. We further suggest that this ABC-transporter in R. capsulatus is involved in the formation and export of polysulfide species to the periplasm.

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.

Effect of Oxidation and Protonation States on [2Fe-2S] Cluster Nitrosylation Giving {Fe(NO)2}9 Dinitrosyl Iron Complexes (DNICs).

The nitrosylation of biological Fe/S clusters to give protein-bound dinitrosyl iron complexes (DNICs) is physiologically important. Biomimetic studies on the reaction of synthetic [2Fe-2S] clusters with NO have so far been limited to diferric model complexes. This work now compares the nitrosylation of [2Fe-2S] clusters with SN- or NN-chelating benzimidazolate/thiophenolate or bis(benzimidazolate) capping ligands in their diferric (12- and 22-) and mixed-valent (FeIIFeIII, 13-, and 23-) forms. Furthermore, the effect of protonation of the imidazole part of the SN ligand has been probed on both the nitrosylation reaction and properties of the resulting DNIC. The reaction of 12- and 22- with 4 equiv NO yields the new anionic {Fe(NO)2}9 DNICs 3- and 4-, respectively, which have been comprehensively characterized, including X-ray crystallography of their PPN+ salts. Nitrosylation of mixed-valent [2Fe-2S] clusters 13- and 23- first leads to slow oxidation to the corresponding diferric congeners, followed by core degradation and DNIC formation. In the case of 23-, a second diferric intermediate very similar to 22- is detected by UV-vis spectroscopy, but could not be further identified. Nitrosylation of 1H2 gives the neutral, N-protonated DNIC 3H, and acid/base titrations show that interconversion between 3- and 3H is reversible. Peripheral ligand protonation leads to a blue shift of the NO stretching vibrations by about 23 cm-1 and a significant shift of the reduction potential to less negative values (Δ E1/2 = 0.26 V), but no effect on 57Fe Mössbauer parameters is observed. Density functional theory calculations based on the structure of 3- indicate that the electronic ground-state properties of 3- and 3H are similar, although the NO(π*) → Fe 3d π-donation is slightly increased and π-backbonding is slightly decreased upon protonation. As a result, protonation has a significant effect on the NO stretching frequencies, but only minor effects on the Fe-(NO)2 modes. This is confirmed by nuclear inelastic scattering of 3- and 3H, which shows no clear influence of protonation on the energy of the Fe-(NO)2 bending and stretching modes occurring in the range 400-600 cm-1, but characteristic changes below 350 cm-1 that reflect perturbation of free rotary motion of the thiophenolate and benzimidazole ring systems of the capping ligand after N-protonation. These findings add to the understanding of [2Fe-2S] cluster nitrosylation and will help to identify DNICs resulting from the reaction of NO with Fe/S cofactors featuring alternative, proton-responsive histidine ligands such as the Rieske and mitoNEET [2Fe-2S] clusters.