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2009 Annual Science Report

Montana State University Reporting  |  JUL 2008 – AUG 2009

Structure, Function, and Biosynthesis of the Complex Iron-Sulfur Clusters at the Active Sites of Nitrogenases and Hydrogenases

Project Summary

Iron-sulfur clusters are thought to be among the most ancient cofactors in living systems. The iron-sulfur enzyme thrust is focused on examining the structure, mechanism, and biosynthesis of the complex Fe-S enzymes nitrogenase and hydrogenase. Biochemical, biophysical, and structure biology approaches are being employed to provide insights into complex iron-sulfur biosynthesis to establish paradigms for complex iron-sulfur cluster biosynthesis that can be placed in the context of the evolution of iron-sulfur motifs from the abiotic to biotic systems.

4 Institutions
3 Teams
9 Publications
1 Field Site
Field Sites

Project Progress

During the current funding period we have made significant progress in the area of [FeFe]-hydrogenase H cluster biosynthesis. We have determined that the complex H cluster consisting of a [4Fe-4S] cluster bridged to a unique 2Fe subcluster with unique diatomic (carbon monoxide and cyanide) and dithiolate ligands is synthesized in a stepwise manner. Our results indicate that the [4Fe-4S] cluster moiety is synthesized by generalized host cell machinery in the same manner that [4Fe-4S] clusters involved in various electron transfer reactions in cells are synthesized. The unique 2Fe subcluster synthesis and insertion follows [4Fe-4S] cluster synthesis and insertion and requires specialized biosynthesis machinery unique to [FeFe]-hydrogenases. The 2Fe subcluster synthesis requires two radical SAM enzymes that couple radical chemistry to the production of the non protein ligands and occurs on a scaffold that we have identified as the HydF gene product much in the same manner is the scaffold dependent synthesis of the nitrogenase FeMo-cofactor. We have identified a potential link between this process and the biosynthesis of the evolutionarily unrelated [Fe]-hydrogenases that also possess diatomic non-protein carbon monoxide ligands. Our recent results indicate that a radical SAM enzyme termed HmdB is involved in the mononuclear Hmd hydrogenase active site biosynthesis. The substrates for these radical SAM enzymes or the metabolic sources of the non protein ligands in these systems remain elusive and we are employing a variety of approaches including mass spectrometry to solve this challenging problem. Our most recent results involve the elucidation of the structure of the [FeFe]-hydrogenase expressed in a background devoid of the 2Fe subcluster biosynthetic machinery, that provide significant insights into the mechanism of 2Fe subcluster insertion, the final stage in [FeFe]-hydrogenase maturation and establish fundamental parallels between H-cluster biosynthesis and nitrogenase FeMo-cofactor biosynthesis.

Hypothetical Scheme for [FeFe]-hydrogenase Maturation

In two steps, radical-SAM enzymes HydE and HydG use HydF as a
scaffold to modify a basic [2Fe-2S] cluster with the addition of (1) a
dithiolate ligand and (2) CO and CN- ligands. The first step is
proposed to take place via a sulfur-carbon bond insertion reaction
and the second step via an amino acid precursor by radical formation.
The magenta atoms of the 2Fe subcluster represent
hypothetical protein ligands. After assembly of the ligand-modified
2Fe subcluster, HydF* transfers it to HydAΔEFG, which already
houses the [4Fe-4S] subcluster of the H-cluster, completing activation
of [FeFe]-hydrogenase and H-cluster biosynthesis. An atomic model
of the H-cluster in activated HydAΔEFG is depicted showing the
coupling of the [4Fe-4S] cubane to the 2Fe subcluster with CO,CN-,
and dithiolate ligands via a bridging cysteine ligand [from Protein
Data Bank entry 3C8Y]; the coloring scheme is as follows: dark
red for Fe, orange for S, red for O, blue for N, dark gray for C, and
magenta for an unknown atom of dithiolate ligand. Also, a water
molecule is present at the distal Fe of the 2Fe subcluster in the
presumed oxidized state of the H-cluster. The homology models of
HydAΔEFG and HydA of C. reinhardtii were constructed using the
homology server Phyre, and HydA from C. reinhardtii was
threaded on HydA from Cl. pasteurianum (CpI) during sequence
alignment. Ribbon representations of the structures were made in
PyMOL with the C-terminal domain colored red.

Publications

  • Boyd, E. S., Spear, J. R., & Peters, J. W. (2009). [FeFe] Hydrogenase Genetic Diversity Provides Insight into Molecular Adaptation in a Saline Microbial Mat Community. Applied and Environmental Microbiology, 75(13), 4620–4623. doi:10.1128/aem.00582-09

  • Chandra, T., Silver, S. C., Zilinskas, E., Shepard, E. M., Broderick, W. E., & Broderick, J. B. (2009). Spore Photoproduct Lyase Catalyzes Specific Repair of the 5 R but Not the 5 S Spore Photoproduct. Journal of the American Chemical Society, 131(7), 2420–2421. doi:10.1021/ja807375c

  • McGlynn, S. E., Boyd, E. S., Shepard, E. M., Lange, R. K., Gerlach, R., Broderick, J. B., & Peters, J. W. (2009). Identification and Characterization of a Novel Member of the Radical AdoMet Enzyme Superfamily and Implications for the Biosynthesis of the Hmd Hydrogenase Active Site Cofactor. Journal of Bacteriology, 192(2), 595–598. doi:10.1128/jb.01125-09

  • McGlynn, S. E., Mulder, D. W., Shepard, E. M., Broderick, J. B., & Peters, J. W. (2009). Hydrogenase cluster biosynthesis: organometallic chemistry nature’s way. Dalton Trans., None(22), 4274. doi:10.1039/b821432h

  • McGlynn, S. E., Shepard, E. M., Winslow, M. A., Naumov, A. V., Duschene, K. S., Posewitz, M. C., … Peters, J. W. (2008). HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis. FEBS Letters, 582(15), 2183–2187. doi:10.1016/j.febslet.2008.04.063

  • Mulder, D. W., Boyd, E. S., Sarma, R., Lange, R. K., Endrizzi, J. A., Broderick, J. B., & Peters, J. W. (2010). Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG. Nature, 465(7295), 248–251. doi:10.1038/nature08993

  • Mulder, D. W., Ortillo, D. O., Gardenghi, D. J., Naumov, A. V., Ruebush, S. S., Szilagyi, R. K., … Peters, J. W. (2009). Activation of HydA ΔEFG Requires a Preformed [4Fe-4S] Cluster. Biochemistry, 48(26), 6240–6248. doi:10.1021/bi9000563

  • Sarma, R., Barney, B. M., Hamilton, T. L., Jones, A., Seefeldt, L. C., & Peters, J. W. (2008). Crystal Structure of the L Protein of Rhodobacter sphaeroides Light-Independent Protochlorophyllide Reductase with MgADP Bound: A Homologue of the Nitrogenase Fe Protein † ‡. Biochemistry, 47(49), 13004–13015. doi:10.1021/bi801058r

  • Shepard, E.M. & Broderick, J.B.  (2009).  S-Adenosylmethionine and iron-sulfur clusters in biological radical reactions: The radical SAM superfamily [Book Chapter].  Comprehensive Natural Products Chemistry II.  Vol. In press.
  • PROJECT INVESTIGATORS:
    Joan Broderick Joan Broderick
    Project Investigator
    John Peters John Peters
    Project Investigator
  • PROJECT MEMBERS:
    Robert Szilagyi
    Co-Investigator

    Alexios Grigoropoulos
    Postdoc

    Eric Shepard
    Postdoc

    Alexandra Bueling
    Graduate Student

    Ben Duffus
    Graduate Student

    Shawn McGlynn
    Graduate Student

    David Mulder
    Graduate Student

    Paul Jordan
    Undergraduate Student

  • RELATED OBJECTIVES:
    Objective 3.1
    Sources of prebiotic materials and catalysts

    Objective 3.2
    Origins and evolution of functional biomolecules

    Objective 3.3
    Origins of energy transduction

    Objective 6.1
    Effects of environmental changes on microbial ecosystems

    Objective 6.2
    Adaptation and evolution of life beyond Earth

    Objective 7.1
    Biosignatures to be sought in Solar System materials

    Objective 7.2
    Biosignatures to be sought in nearby planetary systems