Structural consequences of turnover-induced homocitrate loss in nitrogenase
Abstract
Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein – a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA – that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.
Additional Information
© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. This work was funded by support from the Howard Hughes Medical Institute (D.C.R.), NIH grant GM045162 (D.C.R.), and NIH GM143836-01 (R.A.W.). The foundational contributions of Dr. Thomas Spatzal to establishing the anaerobic Vitrobot system are gratefully acknowledged. We thank Dr. Jens Kaiser, Dr. Songye Chen, Dr. Trixia Buscagan, and Przemyslaw Dutka for their invaluable discussions. IC-MS and ICP-MS were performed on instrumentation made available by the Resnick Sustainability Institute's Water and Environment Lab at the California Institute of Technology. EPR data were collected at the Caltech EPR Facility which is supported by NSF-1531940. Bottom-up mass spectrometry of protein samples was performed at the Beckman Proteome Exploration Laboratory supported by the Arnold and Mabel Beckman Foundation. We thank Dr. Nathan Dalleska, Dr. Paul Oyala, and Dr. Ting-Yu Wang for their support and assistance with these analyses. The generous support of the Beckman Institute for the Caltech CryoEM Resource Center was essential for the performance of this research. We dedicate this work to our co-author Dr. James B. Howard who passed during the course of this work. Contributions. R.A.W. and D.C.R. designed experiments. R.A.W. performed sample preparation, biochemical analyses, electron microscopy data collection and analyses, model building, and refinement. A.O.M. performed cryoEM model building and refinement. A.O. purified protein from the mutant A. vinelandii. B.B.W. performed initial tests of anaerobic cryoEM workflow. J.B.H. provided valuable advice on the project and the manuscript. D.C.R. supervised all research. R.A.W. and D.C.R. wrote the manuscript and all authors contributed to revisions. Data availability. The single particle cryoEM maps and models generated in this study have been deposited into the PDB and EMDB for release upon publication. Reconstructed maps and refined models have been deposited with the following PDB and EMDB codes: 8CRS, EMD-26957 (MoFe^(As-isolated)); PDB 8DBX, EMD-27316 (MoFe^(Oxidized)); PDB 8ENL, EMD-28272 (MoFe^(Alkaline-inactivated)); PDB 8ENM, EMD-28273 (MoFe^(Alkaline)); PDB 8ENN, EMD-28274 (MoFe^(ΔNifV)); PDB 8ENO, EMD-28275 (MoFe^(ΔNifV)-NafT). The Uniprot all-reviewed A. vinelandii database was used for the analysis of mass spectrometry data. All other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper. The authors declare no competing interests.Attached Files
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Supplemental Material - 41467_2023_36636_MOESM1_ESM.pdf
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Supplemental Material - 41467_2023_36636_MOESM4_ESM.xls
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Additional details
- PMCID
- PMC9968304
- Eprint ID
- 122084
- Resolver ID
- CaltechAUTHORS:20230630-537337000.8
- Howard Hughes Medical Institute (HHMI)
- GM045162
- NIH
- GM143836-01
- NIH
- CHE-1531940
- NSF
- Arnold and Mabel Beckman Foundation
- Caltech Beckman Institute
- Created
-
2023-07-01Created from EPrint's datestamp field
- Updated
-
2023-07-01Created from EPrint's last_modified field
- Caltech groups
- Resnick Sustainability Institute