Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
13 May 2024
13 May 2024
Historique:
received:
28
06
2023
accepted:
25
04
2024
medline:
14
5
2024
pubmed:
14
5
2024
entrez:
13
5
2024
Statut:
epublish
Résumé
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 E
Identifiants
pubmed: 38740794
doi: 10.1038/s41467-024-48271-8
pii: 10.1038/s41467-024-48271-8
doi:
Substances chimiques
Nitrogenase
EC 1.18.6.1
Molybdoferredoxin
0
Selenium
H6241UJ22B
Sulfur
70FD1KFU70
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
4041Subventions
Organisme : U.S. Department of Health & Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)
ID : GM045162
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : ID311061829
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : ID 235777276
Informations de copyright
© 2024. The Author(s).
Références
Boyd, E. S. & Peters, J. W. New insights into the evolutionary history of biological nitrogen fixation. Front. Microbiol. 4, 1–12 (2013).
doi: 10.3389/fmicb.2013.00201
Hoffman, B. M., Lukoyanov, D., Yang, Z.-Y., Dean, D. R. & Seefeldt, L. C. Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062 (2014).
pubmed: 24467365
pmcid: 4012840
doi: 10.1021/cr400641x
Einsle, O. & Rees, D. C. Structural enzymology of nitrogenase enzymes. Chem. Rev. 120, 4969–5004 (2020).
pubmed: 32538623
pmcid: 8606229
doi: 10.1021/acs.chemrev.0c00067
Kim, J. S. & Rees, D. C. Crystallographic structure and functional implications of the nitrogenase molybdenum Iron protein from Azotobacter vinelandii. Nature 360, 553–560 (1992).
pubmed: 25989647
doi: 10.1038/360553a0
Einsle, O. et al. Nitrogenase MoFe-protein at 1.16 Å resolution: a central ligand in the FeMo-cofactor. Science 297, 1696–1700 (2002).
pubmed: 12215645
doi: 10.1126/science.1073877
Spatzal, T. et al. Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334, 940 (2011).
pubmed: 22096190
pmcid: 3268367
doi: 10.1126/science.1214025
Seefeldt, L. C., Hoffman, B. M. & Dean, D. R. Electron transfer in nitrogenase catalysis. Curr. Opin. Chem. Biol. 16, 19–25 (2012).
pubmed: 22397885
pmcid: 3328587
doi: 10.1016/j.cbpa.2012.02.012
Rutledge, H. L. & Tezcan, F. A. Electron transfer in nitrogenase. Chem. Rev. 120, 5158–5193 (2020).
pubmed: 31999100
pmcid: 7466952
doi: 10.1021/acs.chemrev.9b00663
Hageman, R. V. & Burris, R. H. Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii. Biochemistry 17, 4117–4124 (1978).
pubmed: 708696
doi: 10.1021/bi00613a002
Milton, R. D. & Minteer, S. D. Nitrogenase bioelectrochemistry for synthesis applications. Acc. Chem. Res. 52, 3351–3360 (2019).
pubmed: 31800207
doi: 10.1021/acs.accounts.9b00494
Lowe, D. J. & Thorneley, R. N. F. The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N
doi: 10.1042/bj2240895
Hoffman, B. M., Lukoyanov, D., Dean, D. R. & Seefeldt, L. C. Nitrogenase: a draft mechanism. Acc. Chem. Res. 46, 587–595 (2013).
pubmed: 23289741
pmcid: 3578145
doi: 10.1021/ar300267m
Hoeke, V. et al. High-resolution ENDOR spectroscopy combined with quantum chemical calculations reveals the structure of nitrogenase Janus intermediate E
pubmed: 31310109
pmcid: 6956989
doi: 10.1021/jacs.9b04474
Lukoyanov, D. et al. Hydride conformers of the nitrogenase FeMo-cofactor two-electron reduced state E
pubmed: 29575898
pmcid: 6008734
doi: 10.1021/acs.inorgchem.8b00271
Lukoyanov, D. et al. Reductive elimination of H
pubmed: 27529724
pmcid: 5024552
doi: 10.1021/jacs.6b06362
Lukoyanov, D. et al. ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N
pubmed: 21744838
pmcid: 3156091
doi: 10.1021/ja2036018
Cao, L. & Ryde, U. Putative reaction mechanism of nitrogenase after dissociation of a sulfide ligand. J. Catal. 391, 247–259 (2020).
doi: 10.1016/j.jcat.2020.08.028
Rawlings, J. et al. Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence. J. Biol. Chem. 253, 1001–1004 (1978).
pubmed: 203578
doi: 10.1016/S0021-9258(17)38102-4
Venters, R. A. et al. ENDOR of the resting state of nitrogenase molybdenum–iron proteins of Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianum:
doi: 10.1021/ja00272a054
Goldberg, D. P. et al. EPR spectra from “EPR-silent” species: High-field EPR spectroscopy of manganese(III) porphyrins. J. Am. Chem. Soc. 119, 8722–8723 (1997).
doi: 10.1021/ja971169o
Telser, J. EPR interactions – Zero-field splittings. eMagRes 6, 207–233 (2017).
doi: 10.1002/9780470034590.emrstm1501
Fisher, K. et al. Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function. J. Inorg. Biochem. 101, 1649–1656 (2007).
pubmed: 17845818
doi: 10.1016/j.jinorgbio.2007.07.037
Fisher, K., Newton, W. E. & Lowe, D. J. Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: Evidence for S=3/2 spin states from reduced MoFe-protein intermediates. Biochemistry 40, 3333–3339 (2001).
pubmed: 11258953
doi: 10.1021/bi0012686
Shaw, S. et al. Nitrite and hydroxylamine as nitrogenase substrates: Mechanistic implications for the pathway of N
pubmed: 25136926
pmcid: 4160268
doi: 10.1021/ja507123d
Benton, P. M. C. et al. Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: Trapping propargyl alcohol with an α−70-substituted MoFe protein. Biochemistry 42, 9102–9109 (2003).
pubmed: 12885243
doi: 10.1021/bi034595x
Spatzal, T., Perez, K. A., Einsle, O., Howard, J. B. & Rees, D. C. Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase. Science 345, 1620–1623 (2014).
pubmed: 25258081
pmcid: 4205161
doi: 10.1126/science.1256679
Rohde, M., Grunau, K. & Einsle, O. CO binding to the FeV cofactor of CO-reducing vanadium nitrogenase at atomic resolution. Angew. Chem. Int. Ed. 59, 23626–23630 (2020).
doi: 10.1002/anie.202010790
Spatzal, T., Perez, K. A., Howard, J. B. & Rees, D. C. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor. eLife 4, e11620 (2015).
pubmed: 26673079
pmcid: 4755756
doi: 10.7554/eLife.11620
Sippel, D. et al. A bound reaction intermediate sheds light on the mechanism of nitrogenase. Science 359, 1484–1489 (2018).
pubmed: 29599235
doi: 10.1126/science.aar2765
Henthorn, J. T. et al. Localized electronic structure of nitrogenase FeMoco revealed by selenium K-edge high resolution X-ray absorption spectroscopy. J. Am. Chem. Soc. 141, 13676–13688 (2019).
pubmed: 31356071
pmcid: 6716209
doi: 10.1021/jacs.9b06988
Spatzal, T. et al. Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement. Nat. Commun. 7, 10902 (2016).
pubmed: 26973151
pmcid: 4793075
doi: 10.1038/ncomms10902
Chiang, Y.-W., Borbat, P. P. & Freed, J. H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–295 (2005).
pubmed: 15649755
doi: 10.1016/j.jmr.2004.10.012
Jeschke, G. et al. DeerAnalysis2006 – a comprehensive software package for analyzing pulsed ELDOR data. Appl. Magn. Reson. 30, 473–498 (2006).
doi: 10.1007/BF03166213
Rein, S., Lewe, P., Andrade, S. L., Kacprzak, S. & Weber, S. Global analysis of complex PELDOR time traces. J. Magn. Reson. 295, 17–26 (2018).
pubmed: 30092553
doi: 10.1016/j.jmr.2018.07.015
Edwards, T. H. & Stoll, S. Optimal Tikhonov regularization for DEER spectroscopy. J. Magn. Reson. 288, 58–68 (2018).
pubmed: 29414064
pmcid: 5840305
doi: 10.1016/j.jmr.2018.01.021
Ibáñez, L. F. & Jeschke, G. General regularization framework for DEER spectroscopy. J. Magn. Reson. 300, 28–40 (2019).
doi: 10.1016/j.jmr.2019.01.008
Azarkh, M. & Groenen, E. J. J. Simulation of multi-frequency EPR spectra for a distribution of the zero-field splitting. J. Magn. Reson. 255, 106–113 (2015).
pubmed: 25955436
doi: 10.1016/j.jmr.2015.04.006
George, G. N., Prince, R. C. & Bare, R. E. Electron paramagnetic resonance spectroscopy of the iron−molybdenum cofactor of Clostridium pasteurianum nitrogenase. Inorg. Chem. 35, 434–438 (1996).
pubmed: 11666225
doi: 10.1021/ic950740m
Hagen, W. R. Wide zero field interaction distributions in the high-spin EPR of metalloproteins. Mol. Phys. 105, 2031–2039 (2007).
doi: 10.1080/00268970701558570
Hagen, W. R., Hearshen, D. O., Harding, L. J. & Dunham, W. R. Quantitative numerical analysis of g strain in the EPR of distributed systems and its importance for multicenter metalloproteins. J. Magn. Reson. 61, 233–244 (1985).
Hearshen, D. O. et al. An analysis of g strain in the EPR of two [2Fe-2S] ferredoxins. evidence for a protein rigidity model. J. Magnet. Resonance 69, 440–459 (1986). .
Froncisz, W. & Hyde, J. S. Broadening by strains of lines in the g‐parallel region of Cu
doi: 10.1063/1.440548
Lee, H.-I. et al. The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM show it is not an exchangeable nitrogen. J. Am. Chem. Soc. 125, 5604–5605 (2003).
pubmed: 12733878
doi: 10.1021/ja034383n
Lee, H.-I., Doan, P. E. & Hoffman, B. M. General analysis of
pubmed: 10479552
doi: 10.1006/jmre.1999.1803
Azarkh, M., Gast, P., Mason, A. B., Groenen, E. J. J. & Mathies, G. Analysis of the EPR spectra of transferrin: the importance of a zero-field-splitting distribution and 4th-order terms. Phys. Chem. Chem. Phys. 21, 16937–16948 (2019).
pubmed: 31339131
doi: 10.1039/C9CP02626F
Nehrkorn, J., Telser, J., Holldack, K., Stoll, S. & Schnegg, A. Simulating frequency-domain electron paramagnetic resonance: bridging the gap between experiment and magnetic parameters for high-spin transition-metal ion complexes. J. Phys. Chem. B 119, 13816–13824 (2015).
pubmed: 26154490
doi: 10.1021/acs.jpcb.5b04156
Morrison, C. N., Spatzal, T. & Rees, D. C. Reversible protonated resting state of the nitrogenase active site. J. Am. Chem. Soc. 139, 10856–10862 (2017).
pubmed: 28692802
pmcid: 5553094
doi: 10.1021/jacs.7b05695
Lukoyanov, D. et al. A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates. Inorg. Chem. 53, 3688–3693 (2014).
pubmed: 24635454
pmcid: 3993915
doi: 10.1021/ic500013c
Chica, B. et al. Defining intermediates of nitrogenase MoFe protein during N
pubmed: 32787260
doi: 10.1021/jacs.0c06343
Lukoyanov, D., Barney, B. M., Dean, D. R., Seefeldt, L. C. & Hoffman, B. M. Connecting nitrogenase intermediates with the kinetic scheme for N
pubmed: 17251348
pmcid: 1785236
doi: 10.1073/pnas.0610975104
Wessjohann, L. A., Schneider, A., Abbas, M. & Brandt, W. Selenium in chemistry and biochemistry in comparison to sulfur. Biol. Chem. 388, 997–1006 (2007).
pubmed: 17937613
doi: 10.1515/BC.2007.138
Zheng, B., Chen, X.-D., Zheng, S.-L. & Holm, R. H. Selenium as a structural surrogate of Sulfur: Template-assisted assembly of five types of Tungsten–Iron–Sulfur/Selenium clusters and the structural fate of chalcogenide reactants. J. Am. Chem. Soc. 134, 6479–6490 (2012).
pubmed: 22424175
pmcid: 3353770
doi: 10.1021/ja3010539
Spiller, N., Chilkuri, V. G., DeBeer, S., Neese, F. & Sulfur, V. S. Selenium as bridging ligand in di-iron complexes: A theoretical analysis. Eur. J. Inorg. Chem. 2020, 1525–1538 (2020).
doi: 10.1002/ejic.202000033
Arias, R. J. Examination of selenium incorporation and product formation in the nitrogenase FeMo-cofactor. Ph.D thesis, Califorina Institute of Technology Pasadena California (USA) (2018).
Dance, I. Mechanisms of the S/CO/Se interchange reactions at FeMo-co, the active site cluster of nitrogenase. Dalton Trans. 45, 14285–14300 (2016).
pubmed: 27534727
doi: 10.1039/C6DT03159E
Trncik, C., Detemple, F. & Einsle, O. Iron-only Fe-Nitrogenase underscores common catalytic principles in biological Nitrogen fixation. Nat. Catal. 6, 415–424 (2023).
doi: 10.1038/s41929-023-00952-1
Lee, H.-I., Cameron, L. M., Hales, B. J. & Hoffman, B. M. CO binding to the FeMo Cofactor of CO-inhibited nitrogenase:
doi: 10.1021/ja9715096
Tikhonov, A. N. Solution of incorrectly formulated problems and the regularization method. Sov. Math. Dokl. 4, 1035–1038 (1963).
Vucelic, M. & Mijovic, S. Regularization method and applications in spectroscopy. J. Quant. Spectrosc. Radiat. Transf. 56, 617–621 (1996).
doi: 10.1016/0022-4073(96)00085-4
Tozzo, V. et al. Where do we stand in regularization for life science studies? J. Computational Biol. 29, 213–232 (2022).
doi: 10.1089/cmb.2019.0371
Taabazuing, C. Y., Fermann, J., Garman, S. & Knapp, M. J. Substrate promotes productive gas binding in the α-ketoglutarate-dependent oxygenase FIH. Biochemistry 55, 277–286 (2016).
pubmed: 26727884
doi: 10.1021/acs.biochem.5b01003
Gaffney, B. J. EPR of mononuclear non-heme Iron proteins. Biol. Magn. Reson. 28, 233–268 (2009).
pubmed: 20428459
pmcid: 2860145
doi: 10.1007/978-0-387-84856-3_6
Corry, T. A. & O’Malley, P. J. Molecular identification of a high-spin deprotonated intermediate during the S
pubmed: 32431144
doi: 10.1021/jacs.0c01351
Hagen, W. R. & Louro, R. O. A comparative multi-frequency EPR Study of dipolar interaction in tetra-heme Cytochromes. Int. J. Mol. Sci. 24, 12713 (2023).
pubmed: 37628894
pmcid: 10454114
doi: 10.3390/ijms241612713
Sato, M., Kon, H., Kumaki, K. & Nebert, D. W. Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character. Biochimica et. Biophysica Acta (BBA) - Gen. Subj. 498, 403–421 (1977).
doi: 10.1016/0304-4165(77)90279-3
Ge, N. et al. Rationalization of single-molecule magnet behavior in a three-coordinate Fe(III) complex with a high-spin state (S = 5/2). Inorg. Chem. Front. 5, 2486–2492 (2018).
doi: 10.1039/C8QI00701B
Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).
pubmed: 16188474
doi: 10.1016/j.jmr.2005.08.013
Stoll, S. & Britt, R. D. General and efficient simulation of pulse EPR spectra. Phys. Chem. Chem. Phys. 11, 6614–6625 (2009).
pubmed: 19639136
doi: 10.1039/b907277b