The Thioredoxin System Reduces Protein Persulfide Intermediates Formed during the Synthesis of Thio-Cofactors in Bacillus subtilis.

The biosynthesis of Fe-S clusters and other thio-cofactors requires the participation of redox agents. A shared feature in these pathways is the formation of transient protein persulfides, which are susceptible to reduction by artificial reducing agents commonly used in reactions in vitro. These agents modulate the reactivity and catalytic efficiency of biosynthetic reactions and, in some cases, skew the enzymes' kinetic behavior, bypassing sulfur acceptors known to be critical for the functionality of these pathways in vivo. Here, we provide kinetic evidence for the selective reactivity of the Bacillus subtilis Trx (thioredoxin) system toward protein-bound persulfide intermediates. Our results demonstrate that the redox flux of the Trx system modulates the rate of sulfide production in cysteine desulfurase assays. Likewise, the activity of the Trx system is dependent on the rate of persulfide formation, suggesting the occurrence of coupled reaction schemes between both enzymatic systems in vitro. Inactivation of TrxA (thioredoxin) or TrxR (thioredoxin reductase) impairs the activity of Fe-S enzymes in B. subtilis, indicating the involvement of the Trx system in Fe-S cluster metabolism. Surprisingly, biochemical characterization of TrxA reveals that this enzyme is able to coordinate Fe-S species, resulting in the loss of its reductase activity. The inactivation of TrxA through the coordination of a labile cluster, combined with its proposed role as a physiological reducing agent in sulfur transfer pathways, suggests a model for redox regulation. These findings provide a potential link between redox regulation and Fe-S metabolism.

Metallocluster transactions: dynamic protein interactions guide the biosynthesis of Fe-S clusters in bacteria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors present in all domains of life. The chemistries catalyzed by these inorganic cofactors are diverse and their associated enzymes are involved in many cellular processes. Despite the wide range of structures reported for Fe-S clusters inserted into proteins, the biological synthesis of all Fe-S clusters starts with the assembly of simple units of 2Fe-2S and 4Fe-4S clusters. Several systems have been associated with the formation of Fe-S clusters in bacteria with varying phylogenetic origins and number of biosynthetic and regulatory components. All systems, however, construct Fe-S clusters through a similar biosynthetic scheme involving three main steps: (1) sulfur activation by a cysteine desulfurase, (2) cluster assembly by a scaffold protein, and (3) guided delivery of Fe-S units to either final acceptors or biosynthetic enzymes involved in the formation of complex metalloclusters. Another unifying feature on the biological formation of Fe-S clusters in bacteria is that these systems are tightly regulated by a network of protein interactions. Thus, the formation of transient protein complexes among biosynthetic components allows for the direct transfer of reactive sulfur and Fe-S intermediates preventing oxygen damage and reactions with non-physiological targets. Recent studies revealed the importance of reciprocal signature sequence motifs that enable specific protein-protein interactions and consequently guide the transactions between physiological donors and acceptors. Such findings provide insights into strategies used by bacteria to regulate the flow of reactive intermediates and provide protein barcodes to uncover yet-unidentified cellular components involved in Fe-S metabolism.

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions.

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes.