Structural characterization of the P1 intermediate state of the P-cluster of nitrogenase

Stephen M. Keable; Oleg A. Zadvornyy; Lewis E. Johnson; Bojana Ginovska; Andrew J. Rasmussen; Karamatullah Danyal; Brian J. Eilers; Gregory A. Prussia; Axl X. LeVan; Simone Raugei; Lance C. Seefeldt; John W. Peters

doi:10.1074/jbc.RA118.002435

Structural characterization of the P¹ intermediate state of the P-cluster of nitrogenase

Stephen M. Keable
, Oleg A. Zadvornyy
, Lewis E. Johnson
, Bojana Ginovska
, Andrew J. Rasmussen
, Karamatullah Danyal
, Brian J. Eilers
, Gregory A. Prussia
, Axl X. LeVan
, Simone Raugei
, Lance C. Seefeldt
, John W. Peters

Research output: Contribution to journal › Article › peer-review

51 Scopus citations

Abstract

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N₂) to ammonia (NH₃) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N₂ is reduced to NH₃. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (P^N) state to the two-electron oxidized P² state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P², P¹, and P^N) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P¹ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P² states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

Original language	English
Pages (from-to)	9629-9635
Number of pages	7
Journal	Journal of Biological Chemistry
Volume	293
Issue number	25
DOIs	https://doi.org/10.1074/jbc.RA118.002435
State	Published - Jun 22 2018
Externally published	Yes

Funding

This work was supported by National Science Foundation Grant MCB-1330807 (to J. W. P. and L. C. S.) and by the United States Department of Energy, Office of Science, Basic Energy Sciences, Division of Chemical Sci-ences, Geosciences, and Biosciences under Contract DE-AC05-76RL01830 (to L. E. J., B. G., and S. R.). The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgments—Use of the Stanford Synchrotron Radiation Light-source (SSRL), SLAC National Accelerator Laboratory, is supported by the United States Department of Energy (DOE), Office of Science, Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393). Computer resources were provided by the W. R. Wiley Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility, located at Pacific Northwest National Laboratory and sponsored by DOE’s Office of Biological and Environmental Research. This work was supported by National Science Foundation Grant MCB-1330807 (to J. W. P. and L. C. S.) and by the United States Department of Energy, Office of Science, Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Contract DE-AC05-76RL01830 (to L. E. J., B. G., and S. R.). The authors declare that they have no conflicts of interest with the contents of this article. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the United States Department of Energy (DOE), Office of Science, Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393). Computer resources were provided by the W. R. Wiley Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility, located at Pacific Northwest National Laboratory and sponsored by DOE?s Office of Biological and Environmental Research. This work was supported by National Science Foundation Grant MCB-1330807 (to J. W. P. and L. C. S.) and by the United States Department of Energy, Office of Science, Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Contract DE-AC05-76RL01830 (to L. E. J., B. G., and S. R.). The authors declare that they have no conflicts of interest with the contents of this article. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the United States Department of Energy (DOE), Office of Science, Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393). Computer resources were provided by the W. R. Wiley Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility, located at Pacific Northwest National Laboratory and sponsored by DOE’s Office of Biological and Environmental Research.

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Keable, S. M., Zadvornyy, O. A., Johnson, L. E., Ginovska, B., Rasmussen, A. J., Danyal, K., Eilers, B. J., Prussia, G. A., LeVan, A. X., Raugei, S., Seefeldt, L. C., & Peters, J. W. (2018). Structural characterization of the P¹ intermediate state of the P-cluster of nitrogenase. Journal of Biological Chemistry, 293(25), 9629-9635. https://doi.org/10.1074/jbc.RA118.002435

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abstract = "Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2 state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2, P1, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1 state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2 states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.",

author = "Keable, \{Stephen M.\} and Zadvornyy, \{Oleg A.\} and Johnson, \{Lewis E.\} and Bojana Ginovska and Rasmussen, \{Andrew J.\} and Karamatullah Danyal and Eilers, \{Brian J.\} and Prussia, \{Gregory A.\} and LeVan, \{Axl X.\} and Simone Raugei and Seefeldt, \{Lance C.\} and Peters, \{John W.\}",

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AU - Rasmussen, Andrew J.

AU - Danyal, Karamatullah

AU - Eilers, Brian J.

AU - Prussia, Gregory A.

AU - LeVan, Axl X.

AU - Raugei, Simone

AU - Seefeldt, Lance C.

AU - Peters, John W.

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N2 - Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2 state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2, P1, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1 state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2 states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

AB - Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2 state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2, P1, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1 state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2 states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

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Structural characterization of the P¹ intermediate state of the P-cluster of nitrogenase

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