Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra

Creators: Heidinger, Lorenz; Perez, Kathryn; Spatzal, Thomas; Einsle, Oliver; Weber, Stefan; Rees, Douglas C.¹; Schleicher, Erik

1. California Institute of Technology

Abstract

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E₀. Two of these species were preliminary assigned to hydrogenated E₂ states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of ³³S and ⁷⁷Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.

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Acknowledgement

We thank Jon Rittle for kindly preparing K⁷⁷SeCN, and Maximilian Mayländer and Johannes Ruhnke for help with the blue-light EPR experiments. We thank Prof. Dr. Brian Hoffman for providing the EPR data sets of freeze-quenched and light-induced samples. Support from NIH Grant GM045162 and the Howard Hughes Medical Institute to D.C.R. is gratefully acknowledged. O.E. acknowledges support from the German Research Foundation (PP 1927, project ID 311061829, and RTG 1976, project ID 235777276). E.S. acknowledges support by the Hans-Fischer-Gesellschaft. L.H., S.W., and E.S. thank the SIBW/DFG for financing EPR instrumentation that is operated within the MagRes Center of the University of Freiburg.

Funding

Open Access funding enabled and organized by Projekt DEAL.

Contributions

L.H., K.P., T.S., O.E., S.W., D.C.R. and E.S. designed the research and conceived the experiments. K.P. and T.S. prepared all samples. L.H. conducted all EPR and ESEEM experiments. L.H. and E.S. analyzed and interpreted the spectroscopic data. L.H. wrote the simulation routines. The figures were generated by L.H. and E.S. The manuscript was written through the contributions of all authors. All authors have reviewed and approved the manuscript.

Data Availability

All data supporting the findings of this study are available with the paper and its supplementary information files. The raw data can be downloaded from the website: https://freidok.uni-freiburg.de/data/246337. Figure 2 is made from the folder Powersweeps_E580, Fig. 5 is made from the folders 2P_ESEEM and 3P_ESEEM, and Fig. 6 is made from the folder Light_Induced. A detailed description of which data was used for which figure can be downloaded from the website: https://freidok.uni-freiburg.de/data/246957. The PDB entry 4TKU can be downloaded from: https://www.rcsb.org/structure/4TKU. Source data are provided as a Source Data file. Source data are provided in this paper.

Code Availability

All code used in this work can be downloaded from the website: https://freidok.uni-freiburg.de/data/246338.

Supplementary Information

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