More Like this

1. 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)

   Abstract 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. 

   more » « less
2. 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. 

   more » « less
3. [FeFe]‐Hydrogenase: Defined Lysate‐Free Maturation Reveals a Key Role for Lipoyl‐H‐Protein in DTMA Ligand Biosynthesis

   https://doi.org/10.1002/anie.202203413  

   Pagnier, Adrien; Balci, Batuhan; Shepard, Eric M.; Yang, Hao; Warui, Douglas M.; Impano, Stella; Booker, Squire J.; Hoffman, Brian M.; Broderick, William E.; Broderick, Joan B. (April 2022, Angewandte Chemie International Edition)

   Abstract Maturation of [FeFe]‐hydrogenase (HydA) involves synthesis of a CO, CN⁻, and dithiomethylamine (DTMA)‐coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S‐adenosyl‐l‐methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]‐hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl‐lipoyl‐H‐protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H‐cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA. 

   more » « less
4. 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. 

   more » « less
5. Biophysical Modeling of SARS-CoV-2 Assembly: Genome Condensation and Budding

   https://doi.org/10.3390/v14102089  

   Li, Siyu; Zandi, Roya (October 2022, Viruses)

   The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spurred unprecedented and concerted worldwide research to curtail and eradicate this pathogen. SARS-CoV-2 has four structural proteins: Envelope (E), Membrane (M), Nucleocapsid (N), and Spike (S), which self-assemble along with its RNA into the infectious virus by budding from intracellular lipid membranes. In this paper, we develop a model to explore the mechanisms of RNA condensation by structural proteins, protein oligomerization and cellular membrane–protein interactions that control the budding process and the ultimate virus structure. Using molecular dynamics simulations, we have deciphered how the positively charged N proteins interact and condense the very long genomic RNA resulting in its packaging by a lipid envelope decorated with structural proteins inside a host cell. Furthermore, considering the length of RNA and the size of the virus, we find that the intrinsic curvature of M proteins is essential for virus budding. While most current research has focused on the S protein, which is responsible for viral entry, and it has been motivated by the need to develop efficacious vaccines, the development of resistance through mutations in this crucial protein makes it essential to elucidate the details of the viral life cycle to identify other drug targets for future therapy. Our simulations will provide insight into the viral life cycle through the assembly of viral particles de novo and potentially identify therapeutic targets for future drug development. 

   more » « less