Sulfur Administration in Fe–S Cluster Homeostasis

Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity.

STUDY OF FLUID DYNAMICS AND SULFUR MASS TRANSFER BETWEEN STEEL AND SLAG IN A LADLE FURNACE CONSIDERING DRAG AND NON-DRAG FORCES

In this work fluid dynamics and a basic study of the sulfur transfer at the steel/slag interface in the ladle during argon gas agitation was developed. Mass transfer and chemical reaction models coupled with Computational Fluid Dynamics (CFD) were employed. The multiphasic simulation was solved using the Eulerian model considering drag and non-drag forces, and the flow pattern was validated through Particle Image Velocimetry (PIV) technique. The sulfur transfer rate was tracked by two approximations: (1) unidirectional constant rate Mass Transfer Model (MTM), and (2) unidirectional constant rate Mass Transfer Model coupled with Chemical Reaction Model (MTM+CRM) using Arrhenius equation. It was found that including the non-drag forces affects the fluid dynamics structure. Otherwise, the desulfurization rates increase as the argon gas flow rate increases, finding that the MTM model predicts ~15% less sulfur in the steel than the MTM+CRM, whose results were compared with plant measurements reports.

Saccharomyces cerevisiae Rhodanese RDL2 Uses the Arg Residue of the Active-Site Loop for Thiosulfate Decomposition

Persulfide, polysulfide and thiosulfate are examples of sulfane sulfur containing chemicals that play multiple functions in biological systems. Rhodaneses are widely present in all three kingdoms of life, which catalyze sulfur transfer among these sulfane sulfur-containing chemicals. The mechanism of how rhodaneses function is not well understood. Saccharomyces cerevisiae rhodanese 2 (RDL2) is involved in mitochondrial biogenesis and cell cycle control. Herein, we report a 2.47 Å resolution structure of RDL2 co-crystallized with thiosulfate (PDB entry: 6K6R). The presence of an extra sulfur atom Sδ, forming a persulfide bond with the Sγ atom of Cys106, was observed. Distinct from the persulfide groups in GlpE (PDB entry:1GMX) and rhobov (PDB entry:1BOI), the persulfide group of RDL2 is located in a peanut-like pocket of the neutral electrostatic field and is far away from positively charged amino acid residues of its active-site loop, suggesting no interaction between them. This finding suggests that the positively charged amino acid residues are not involved in the stabilization of the persulfide group. Activity assays indicate that the Arg111 of the active-site loop is critical for the sulfane sulfur transfer. In vitro assays indicate that Arg propels the thiosulfate decomposition. Thus, we propose that Arg can offer a hydrogen bond-rich, acidic-like microenvironment in RDL2 in which thiosulfate decomposes to release sulfane sulfur. Thr of the active-site loop of rhodaneses has the same functions as Arg. Our proposal may explain the catalyzing mechanism of rhodaneses.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.