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1. The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

   https://doi.org/10.1038/s41598-023-42400-x  

   Saini, Jasleen; Deere, Thomas M.; Lessner, Daniel J. (September 2023, Scientific Reports)

   Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC inMethanosarcina acetivorans, which contains twosufCBgene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or deletesufC1B1andsufC2B2, respectively. Neither the dual repression ofsufC1B1andsufC2B2nor the deletion of bothsufC1B1andsufC2B2affected the growth ofM. acetivoransunder any conditions tested, including diazotrophy. Interestingly, deletion of onlysufC1B1led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion ofsufC2B2acts as a suppressor mutation in the absence ofsufC1B1. In addition, the deletion ofsufC1B1and/orsufC2B2did not affect the total Fe–S cluster content inM. acetivoranscells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis inM. acetivoransand challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens. 

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2. Iron‐Sulfur Clusters: Biogenesis and Biochemistry

   https://doi.org/10.1002/9783527843596  

   Martin_del_Campo, Julia S; Shaybani, Shervin; Dean, Dennis R; Dos_Santos, Patricia C (September 2025, Wiley)

   Leimkühler, Silke; Schwarz, Günter; Lenz, Oliver; Einsle, Oliver (Ed.)

   The biological synthesis of iron–sulfur (Fe–S) clusters requires dedicated pathways involved in the recruitment and activation of Fe and S for cluster assembly with subsequent transfer of preformed clusters to acceptor proteins. Several pathways have been described that include various numbers and types of biosynthetic components, although all of them share the same basic principles for [Fe–S] cluster formation and delivery to target proteins. The NifUS system was discovered and first described in studies involving the model diazotroph Azotobacter vinelandii . It has a dedicated role in serving as the starting point for the activation of [Fe–S] cluster-containing proteins specifically involved in biological nitrogen fixation. NifS is a pyridoxal-5′-phosphate containing l -cysteine-dependent sulfur transferase that delivers activated sulfur to the three-domain NifU, which not only serves as a scaffold for the construction of [2Fe–2S] and [4Fe–4S] clusters but also participates in their delivery to various target proteins involved in nitrogen fixation. Interestingly, analysis of sequenced genomes reveals that the three-domain NifU and NifU-like encoded proteins are not limited to diazotrophs, suggesting a broader role for this system in [Fe–S] cluster biogenesis in other organisms. The colocalization of adjacent nifU and nifS encoding sequences in most of these genomes also provides a strong indication for the involvement of the NifU–NifS [Fe–S] cluster assembly and delivery toolkit for activation of [Fe–S] cluster-containing proteins in a variety of organisms that do not fix nitrogen. 

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3. Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe–4S] Clusters

   https://doi.org/10.1021/acs.biochem.3c00651  

   Prakash, Divya; Xiong, Jin; Chauhan, Shikha S; Walters, Karim A; Kruse, Hannah; Yennawar, Neela; Golbeck, John H; Guo, Yisong; Ferry, James G (June 2024, Biochemistry)

   Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe−4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe−4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms. 

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4. Methods to Investigate the Kinetic Profile of Cysteine Desulfurases

   https://doi.org/https://doi.org/10.1007/978-1-0716-1605-5_10  

   Addo, M.A.; Edwards, A. E.; Dos Santos, P.C. (July 2021, Methods in molecular biology)

   Dos Santos, P.C. (Ed.)

   Biological iron-sulfur (Fe-S) clusters are essential protein prosthetic groups that promote a range of biochemical reactions. In vivo, these clusters are synthesized by specialized protein machineries involved in sulfur mobilization, cluster assembly, and cluster transfer to their target proteins. Cysteine desulfurases initiate the first step of sulfur activation and mobilization in cluster biosynthetic pathways. The reaction catalyzed by these enzymes involves the abstraction of sulfur from the amino acid l-cysteine, with concomitant formation of alanine. The presence and availability of a sulfur acceptor modulate the sulfurtransferase activity of this class of enzymes by altering their reaction profile and catalytic turnover rate. Herein, we describe two methods used to probe the reaction profile of cysteine desulfurases through quantification of alanine and sulfide production in these reactions. 

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5. Single‐Step Sulfur Insertions into Iron Carbide Carbonyl Clusters: Unlocking the Synthetic Door to FeMoco Analogues

   https://doi.org/10.1002/ange.202011517  

   Joseph, Chris; Cobb, Caitlyn R.; Rose, Michael J. (December 2020, Angewandte Chemie)

   Abstract The one‐step syntheses, X‐ray structures, and spectroscopic characterization of synthetic iron clusters, bearing either inorganic sulfides or thiolate with interstitial carbide motifs, are reported. Treatment of iron carbide carbonyl clusters [Fe_n(μ_n‐C)(CO)_m]^x(n=5,6;m=15,16;x=0,−2) with electrophilic sulfur sources (S₂Cl₂, S₈) results in the formation of several μ₄‐S dimers of clusters, and moreover, iron‐sulfide‐(sulfocarbide) clusters. The core sulfocarbide unit {C−S}⁴⁻serves as a structural model for a proposed intermediate in the radicalS‐adenosyl‐L‐methionine biogenesis of the M‐cluster. Furthermore, the electrophilic sulfur strategy has been extended to provide the first ever thiolato‐iron‐carbide complex: an analogous reaction with toluylsulfenyl chloride affords the cluster [Fe₅(μ₅‐C)(SC₇H₇)(CO)₁₃]⁻. The strategy described herein provides a breakthrough towards developing syntheses of biomimetic iron‐sulfur‐carbide clusters like FeMoco. 

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