- [FeFe]-Hydrogenase has several Fe-S clusters N-terminal domain, one [2Fe-2S]

1. Structural and Functional Studies of Nuclear Associated Lamin 1 (Nar1) Protein, a Homolog of Lower Eukaryotic Algal and Bacterial [FeFe] Hydrogenase Alta Howells, Trinity Hamilton, Eric Boyd, John Peters NASA Astrobiology Biogeocatalysis Research Center and the Department of Chemistry and Biochemistry Montana State University, Bozeman, MT 59717 Email: alta.howells@gmail.com Introduction Nar1 & [FeFe]-Hydrogenase Biochemical Research Many eukaryotes utilize the cytoplasmic iron sulfur assembly system (CIA) for the maturation of nuclear and cytoplasmic iron sulfur containing proteins.  Interestingly, the CIA system has been shown to be linked with oxidative stress response via the component Nuclear Associated Lamin 1 (Nar1) protein, a homolog of bacterial [FeFe]-Hydrogenase. Oxidative stress response regulates the amount of cellular reactive oxygen species (ROS) in the cell.  Defects in the response mechanism can lead to an increase of ROS and in turn cause modifications of amino acids such as the irreversible oxidation of sulflhydryl groups which often results in impaired protein function.  Due to their damaging chemical nature, ROS have been attributed to the onset of Parkinson's disease and other human pathological diseases originating in the central nervous system.  Although important to both the oxidative stress response and the maturation of FeS proteins, little is known of the CIA system.  Therefore, we aim to heterologously express and characterize Nar1 from the green alga Chamydomonasreinhardtii to provide a more complete understanding of the role of Nar1 proteins and how they relate structurally, functionally, and evolutionarily to [FeFe]-hydrogenase.  Furthermore, these studies will enable the determination of the role Nar1 has in FeS cluster biosynthesis, metabolism, and in oxidative stress response. Chlostridiumpasteurianum[FeFe]-Hydrogenase Crystal Structure • Primers were designed to amplify Nar1 gene from C. reinhardtiigenomic DNA. Theseprimers were designed with restriction sites that allow for cloning into an expression plasmid that facilitate facile (PolyHis) affinity-based purification by Immobilized Metal Affinity Chromatography (IMAC) • - [FeFe]-Hydrogenase are involved in the production or oxidation of H2. Found in anaerobic bacteria and lower eukaryotes, [FeFe]-hydrogenasetypically function by combining electrons accumulated during anaerobic fermentation with protons to form hydrogen. The key to the reactivity of [FeFe]-hydrogenaseis the complex active site FeScluster, termed the “H cluster”. • - [FeFe]-hydrogenase have several Fe-S clusters • N-terminal domain: one [2Fe-2S] (magenta) and • one [4Fe-4S] (purple) • Central domain: two [4Fe-4S] (green) • C-terminal domain: the H cluster (blue and red) [5] Complex Fe-S cluster, the “H cluster” active site. Step 2 Step 1 C. Reinhardtiigenomic DNA Agarose gel of PCR amplification of Nar1 Expression Vector • Steps • PCR amplification of the Nar1 gene with designed primers. • Insertion of the gene into the expression vector, pETDuet • Transformation into the E. Coli expression strain Step 3 • The complex FeS cluster of [FeFe]-hydrogenaserequires three enzymes, Hyd E, Hyd F and • Hyd G for maturation of the active site. Without the maturase enzymes, HydA∆EFG, still • has the [4Fe-4S] cluster in the C-terminal domain but lacks the 2Fe subcluster. [5] Function of Nar1 E. Coli Expression Ceills Cytosolic Iron-sulfur Cluster Assembly System of Eukaryotes [FeFe]-Hydrogenase“H cluster” Maturation Scheme • In order to check for expression of Nar1 from E. Coli, a western blot was performed using antibodies specific for the His-tag. ExPASy proteomic server was used to estimate the molecular weight of Nar1. • M.W. ≈ 49.9 kDa • The western blot shows that Nar1 is being expressed and that it is in the soluble fraction of the cell. [6] • Although the exact biochemical function of Nar1 is not known, it has been shown to have a role in the cytosolic iron-sulfur cluster assembly (CIA) system and in the response to oxidative stress. • In vivo studies with mutant strains of S. cerevisieadepleted of Nar1 showed: • 1) Reduced radioactive 55Fe incorporation into CIA components and Fe-proteins[2] • 2) Decreased cell viability in the presence of 21% oxygen[3] [1] Conserved Cysteine Residues • Lane: • Cell cytosol before induction • Cytosol after induction • Cell pellet before induction • Pellet after induction [4] Phylogenetic Resurrection Nar1 is an FeS Protein Although Nar1 is ubiquitous in lower Eukaryotes, the exact biochemical function is unknown. Phylogeneticreconstruction of [FeFe]-Hydrogenase and Nar1 homologsindicates that these proteins evolved from a common ancestor.. My research is focused on better understanding the structure and function of Nar1 as well as elucidating the function of the Nar1/[FeFe]-hydrogenaseancestor. • Methods: • Infer the ancestral state nucleic acid • sequence using phylogenetics • Clone into the expression vector, and • characterize the structure/function • • Characterize FeS cluster content, and • its sensitivity to O2. [4] • - [FeFe]-Hydrogenase has several Fe-S clusters • N-terminal domain, one [2Fe-2S] • and one [4Fe-4S] • Medial domain, two [4Fe-4S] • C-terminal domain, the H cluster • Maturase enzymes, Hyd E, F and G required • for maturation of the H cluster. • - Conserved loop region found in the C • terminal domain aids in loading the 2Fe • subcluster of the H-cluster • The crystal structure of Nar1 has yet to be determined. However, biochemical and spectroscopic studies suggest Nar1 possesses two [4Fe-4S] clusters, one in the C-terminal domain and one in the N-terminal domain [4]. • Previous biochemical research • Site directed mutagenesis at • conserved cysteine residues • Electron Paramagnetic Resonance • (EPR) studies spectroscopy • 28% amino acid sequence identity • with Chlostridiumpasteurianum • [FeFe]Hydrogenase [4] • The crystal structure of Nar1 has yet to be determined. However, studies suggest Nar1 has two [4Fe-4S] clusters, one in the C-terminal domain and one in the N-terminal domain. Because Eukaryotes do not have the H-cluster maturase enzymes, it is expected that there is no H-cluster in Nar1. • Structural Studies done on Nar1 • Site directed mutagenesis at conserved cysteine residues • EPR studies • Structural modeling using 28% amino acid sequence • identity with Chlostridium pasteurianum [FeFe]-Hydrogenase Phylogenetic Reconstruction of [FeFe]-Hydrogenase and Nar1 Site Directed Mutagenesis at Conserved Cysteine Residues in Nar1 EPR Spectra of Nar1 The Focus of My Research • - The approach… • Clone, express, and characterize Nar1 from Chlamydomonasreinhardtii - a green alga • whose genome also encodes two [ FeFe]-hydrogenase genes • • Use phylogeneticresurrection to generate a recombinant Nar1/[FeFe]-hydrogenase • ancestor for structural and biochemical characterization Acknowledgments Dr. John Peters, Dr. Eric Boyd, Trinity Hamilton and the Chemistry and Biochemistry Department at Montana State University for making my undergraduate research possible. The Hughes Undergraduate Biology Program and the Howard Hughes Medical Institute for funding this presentation and supporting my undergraduate research. GATCC A A GATCC Nar1 Gene Nar1 Gene G G TTCGA TTCGA Citations: [1] Lill, R., and Muhlenhoff, U. (2006) Iron-sulfur protein biogenesis in eukaryotes: components and mechanisms, Annual review of cell and developmental biology22, 457-486. [2] Balk, J., Pierik, A. J., Aguilar Netz, D. J., Muhlenhoff, U., and Lill, R. (2005) Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis, Biochemical Society transactions33, 86-89. [3] Fujii, M., Adachi, N., Shikatani, K., and Ayusawa, D. (2009) [FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematode, Genes Cells14, 457-468. [4] Urzica, E., Pierik, A. J., Muhlenhoff, U., and Lill, R. (2009) The crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1, Biochemistry. [5] Mulder, David M. (2010) Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydADEFG, Nature 245, 248-252. [6] Shepard, EM. (2010) [FeFE] Hydrogenase maturation: Hyd G catalyzaed synthesis of carbon monnoxide, Journal of American Chemistry Society 132, 9247-9249.