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1. 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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2. Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe 4 Se 4 ] Cluster

   https://doi.org/10.1002/ange.202202271  

   Solomon, Joseph B. ; Tanifuji, Kazuki ; Lee, Chi Chung ; Jasniewski, Andrew J. ; Hedman, Britt ; Hodgson, Keith O. ; Hu, Yilin ; Ribbe, Markus W. ( March 2022 , Angewandte Chemie)

   The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe₄S₄] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe₄Se₄] cluster that is used to substitute the [Fe₄S₄] cluster of theAzotobacter vinelandiiFe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe₄Se₄] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation intoAvNifH. Moreover, these studies reveal that the [Fe₄Se₄] cluster inAvNifH already assumes a partial all‐ferrous state ([Fe₄Se₄]⁰) in the presence of dithionite, where its [Fe₄S₄] counterpart inAvNifH exists solely in the reduced state ([Fe₄S₄]¹⁺). Such a discrepancy in the redox properties of theAvNifH‐associated [Fe₄Se₄] and [Fe₄S₄] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen‐substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

    

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3. Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe 4 Se 4 ] Cluster

   https://doi.org/10.1002/anie.202202271  

   Solomon, Joseph B. ; Tanifuji, Kazuki ; Lee, Chi Chung ; Jasniewski, Andrew J. ; Hedman, Britt ; Hodgson, Keith O. ; Hu, Yilin ; Ribbe, Markus W. ( March 2022 , Angewandte Chemie International Edition)

   The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe₄S₄] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe₄Se₄] cluster that is used to substitute the [Fe₄S₄] cluster of theAzotobacter vinelandiiFe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe₄Se₄] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation intoAvNifH. Moreover, these studies reveal that the [Fe₄Se₄] cluster inAvNifH already assumes a partial all‐ferrous state ([Fe₄Se₄]⁰) in the presence of dithionite, where its [Fe₄S₄] counterpart inAvNifH exists solely in the reduced state ([Fe₄S₄]¹⁺). Such a discrepancy in the redox properties of theAvNifH‐associated [Fe₄Se₄] and [Fe₄S₄] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen‐substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

    

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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. Functional characterization of the HMP‐P synthase of Legionella pneumophila (Lpg1565)

   https://doi.org/10.1111/mmi.14622  

   Paxhia, Michael D. ; Swanson, Michele S. ; Downs, Diana M. ( November 2020 , Molecular Microbiology)

   The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway inSaccharomyces cerevisiaeand other yeast. Previous work found thatScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis inLegionella pneumophila. UnlikeScThi5,LpThi5 functioned in vivo inSalmonella entericaunder multiple growth conditions. The proteinLpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage ofLpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function ofLpThi5.

   Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis inLegionella pneumophilaand provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.

    

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