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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)

   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. Functional relationships of three NFU proteins in the biogenesis of chloroplastic iron‐sulfur clusters

   https://doi.org/10.1002/pld3.303  

   Satyanarayan, Manasa B. ; Zhao, Jun ; Zhang, Jessica ; Yu, Fei ; Lu, Yan ( February 2021 , Plant Direct)

   Iron‐sulfur clusters are required in a variety of biological processes. Biogenesis of iron‐sulfur clusters includes assembly of iron‐sulfur clusters on scaffold complexes and transfer of iron‐sulfur clusters to recipient apoproteins by iron‐sulfur carriers, such as nitrogen‐fixation‐subunit‐U (NFU)‐type proteins.Arabidopsis thalianahas three plastid‐targeted NFUs: NFU1, NFU2, and NFU3. We previously discovered thatnfu2^−/−nfu3^−/−mutants are embryo lethal. The lack of viablenfu2^−/−nfu3^−/−mutants posed a serious challenge. To overcome this problem, we characterizednfu2‐1^−/−nfu3‐2^+/‐andnfu2‐1^+/‐nfu3‐2^−/−sesquimutants. Simultaneous loss‐of‐function mutations inNFU2andNFU3have an additive effect on the declines of 4Fe‐4S‐containing PSI core subunits. Consequently, the sesquimutants had much lower PSI and PSII activities, much less chlorophyll, and much smaller plant sizes, thannfu2‐1andnfu3‐2single mutants. These observations are consistent with proposed roles of NFU3 and NFU2 in the biogenesis of chloroplastic 4Fe‐4S. By performing spectroscopic and in vitro reconstitution experiments, we found that NFU1 may act as a carrier for chloroplastic 4Fe‐4S and 3Fe‐4S clusters. In line with this hypothesis, loss‐of‐function mutations inNFU1resulted in significant declines in 4Fe‐4S‐ and 3Fe‐4S‐containing chloroplastic proteins. The declines of PSI activity and 4Fe‐4S‐containing PSI core subunits innfu1mutants indicate that PSI is the main target of NFU1 action. The reductions in 4Fe‐4S‐containing PSI core proteins and PSI activity innfu3‐2,nfu2‐1, andnfu1single mutants suggest that all three plastid‐targeted NFU proteins contribute to the biogenesis of chloroplastic 4Fe‐4S clusters. Although different insertion sites of T‐DNA lines may cause variations in phenotypic results, mutation severity could be an indicator of the relative importance of the gene product. Our results are consistent with the hypothesis that NFU3 contributes more than NFU2 and NFU2 contributes more than NFU1 to the production of 4Fe‐4S‐containing PSI core subunits.

    

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3. Secondary structure analysis of peptides with relevance to iron–sulfur cluster nesting

   https://doi.org/10.1002/jcc.25741  

   Hanscam, Rebecca ; Shepard, Eric M. ; Broderick, Joan B. ; Copié, Valérie ; Szilagyi, Robert K. ( December 2018 , Journal of Computational Chemistry)

   Peptides coordinated to iron–sulfur clusters, referred to as maquettes, represent a synthetic strategy for constructing biomimetic models of iron–sulfur metalloproteins. These maquettes have been successfully employed as building blocks of engineered heme‐containing proteins with electron‐transfer functionality; however, they have yet to be explored in reactivity studies. The concept of iron–sulfur nesting in peptides is a leading hypothesis in Origins‐of‐Life research as a plausible path to bridge the discontinuity between prebiotic chemical transformations and extant enzyme catalysis. Based on past biomimetic and biochemical research, we put forward a mechanism of maquette reconstitution that guides our development of computational tools and methodologies. In this study, we examined a key feature of the first stage of maquette formation, which is the secondary structure of aqueous peptide models using molecular dynamics simulations based on the AMBER99SB empirical force field. We compared and contrasted S…S distances, [2Fe‐2S] and [4Fe‐4S] nests, and peptide conformations via Ramachandran plots for dissolved Cys and Gly amino acids, the CGGCGGC 7‐mer, and the GGCGGGCGGCGGW 16‐mer peptide. Analytical tools were developed for following the evolution of secondary structural features related to [Fe‐S] cluster nesting along 100 ns trajectories. Simulations demonstrated the omnipresence of peptide nests for preformed [2Fe‐2S] clusters; however, [4Fe‐4S] cluster nests were observed only for the 16‐mer peptide with lifetimes of a few nanoseconds. The origin of the [4Fe‐4S] nest and its stability was linked to a “kinked‐ribbon” peptide conformation. Our computational approach lays the foundation for transitioning into subsequent stages of maquette reconstitution, those being the formation of iron ion/iron–sulfur coordinated peptides. © 2018 Wiley Periodicals, Inc.

    

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4. The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea

   https://doi.org/10.1002/1873-3468.13673  

   Zhao, Cuiping ; Lyu, Zhe ; Long, Feng ; Akinyemi, Taiwo ; Manakongtreecheep, Kasidet ; Söll, Dieter ; Whitman, William B. ; Vinyard, David J. ; Liu, Yuchen ( November 2019 , FEBS Letters)

   The nucleotide binding protein 35 (Nbp35)/cytosolic Fe‐S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe‐S cluster assembly scaffold required for the maturation of Fe‐S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe‐S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal hostMethanococcus maripaludiscontains a [4Fe‐4S] cluster that can be transferred to a [4Fe‐4S] apoprotein. Deletion ofmmp0704fromM. maripaludisdoes not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe‐4S] cluster transfer protein in methanogenic archaea.

    

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5. tRNA Modifications as a Readout of S and Fe-S Metabolism

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

   Dos Santos, P.C. (Ed.)

   Iron-Sulfur (Fe-S) clusters function as core prosthetic groups known to modulate the activity of metalloenzymes, act as trafficking vehicles for biological iron and sulfur, and participate in several intersecting metabolic pathways. The formation of these clusters is initiated by a class of enzymes called cysteine desulfurases, whose primary function is to shuttle sulfur from the amino acid l-cysteine to a variety of sulfur transfer proteins involved in Fe-S cluster synthesis as well as in the synthesis of other thiocofactors. Thus, sulfur and Fe-S cluster metabolism are connected through shared enzyme intermediates, and defects in their associated pathways cause a myriad of pleiotropic phenotypes, which are difficult to dissect. Post-transcriptionally modified transfer RNA (tRNA) represents a large class of analytes whose synthesis often requires the coordinated participation of sulfur transfer and Fe-S enzymes. Therefore, these molecules can be used as biologically relevant readouts for cellular Fe and S status. Methods employing LC-MS technology provide a valuable experimental tool to determine the relative levels of tRNA modification in biological samples and, consequently, to assess genetic, nutritional, and environmental factors modulating reactions dependent on Fe-S clusters. Herein, we describe a robust method for extracting RNA and analytically evaluating the degree of Fe-S-dependent and -independent tRNA modifications via an LC-MS platform. 

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