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Identification of a Spin-Coupled Mo (III) in the Nitrogenase Iron

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Identification of a Spin-Coupled Mo (III) in the Nitrogenase Iron Identification of a spin-coupled Mo(III) in the nitrogenase iron-molybdenum cofactor Ragnar Bjornsson, Frederico A. Lima, Thomas Spatzal, Thomas Weyhermueller, Pieter Glatzel, Eckhard Bill, Oliver Einsle, Frank Neese, Serena Debeer To cite this version: Ragnar Bjornsson, Frederico A. Lima, Thomas Spatzal, Thomas Weyhermueller, Pieter Glatzel, et al.. Identification of a spin-coupled Mo(III) in the nitrogenase iron-molybdenum cofactor. Chemical Science , The Royal Society of Chemistry, 2014, 5 (8), pp.3096-3103. 10.1039/c4sc00337c. hal- 01572851 HAL Id: hal-01572851 https://hal.archives-ouvertes.fr/hal-01572851 Submitted on 8 Aug 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue Identification of a spin-coupled Mo(III)inthe nitrogenase iron–molybdenum cofactor† Cite this: Chem. Sci.,2014,5,3096 a a b a Ragnar Bjornsson,‡ Frederico A. Lima,‡§ Thomas Spatzal,{ Thomas Weyhermuller,¨ Pieter Glatzel,c Eckhard Bill,a Oliver Einsle,*b Frank Neese*a and Serena DeBeer*ad Nitrogenase is a complex enzyme that catalyzes the formation of ammonia utilizing a MoFe7S9C cluster. The presence of a central carbon atom was recently revealed, finally completing the atomic level description of the active site. However, important prerequisites for understanding the mechanism – the total charge, metal oxidation states and electronic structure are unknown. Herein we present high-energy resolution fluorescence detected Mo K-edge X-ray absorption spectroscopy of nitrogenase. Comparison to FeMo model complexes of known oxidation state indicates that the Mo in the FeMo cofactor of nitrogenase is best described as Mo(III), in contrast to the universally accepted Mo(IV) assignment. The oxidation state assignment is supported by theoretical calculations, which reveal the presence of an unusual spin- Received 30th January 2014 coupled Mo(III) site. Although so far Mo(III) was not reported to occur in biology the suggestion raises Creative Commons Attribution 3.0 Unported Licence. Accepted 4th April 2014 interesting parallels with the known homogenous Mo catalysts for N2 reduction, where a Mo(III) DOI: 10.1039/c4sc00337c compound is the N2-binding species. It also requires a reassignment of the Fe oxidation states in the www.rsc.org/chemicalscience cofactor. Introduction The molybdenum–iron component (MoFe) of nitrogenase of Azotobacter vinelandii includes two types of complex metal– Nitrogenase is the enzyme responsible for the catalytic reduc- clusters, the “P-cluster” and the FeMo cofactor (FeMoco). The P- tion of dinitrogen (N2) to ammonia. In contrast to the industrial cluster serves as an electron transfer site, while FeMoco is This article is licensed under a – Haber Bosch process that reacts N2 and H2 at high temperature generally agreed to be the site of dinitrogen reduction. The and pressure, the biological reduction proceeds at ambient FeMo cofactor consists of 7 irons, 1 molybdenum, 9 suldes and temperature and pressure using a complex multicomponent an interstitial light atom that was recently identied as carbon Open Access Article. Published on 09 April 2014. Downloaded 12/06/2015 15:51:41. 3,4 protein system, which utilizes eight electrons, eight protons and by XES, ESEEM and high resolution crystallography. With the 16 MgATP molecules.1,2 Unraveling the structural and mecha- identity of the interstitial atom clear, the basic molecular nistic details of how nature activates the strong bond of dini- structure of the cofactor can at last be considered complete. trogen is of fundamental importance and this knowledge could However, many questions remain about the resting form of the aid in the design of better catalysts. enzyme – namely the total charge of the cofactor, the oxidation state distribution and the electronic structure. Yet, this infor- mation is essential for any informed discussion of the molec- ular level mechanism of dinitrogen reduction. It is also aMax-Planck-Institut fur¨ Chemische Energiekonversion, Stistr. 34-36, 45470 Mulheim¨ essential knowledge for any rationally designed, bio-inspired an der Ruhr, Germany. E-mail: [email protected] catalysts. bInstitute for Biochemistry, Albert-Ludwigs-Universitat¨ Freiburg, Albertstrasse 21, 79104 Freiburg, Germany Presently, three oxidation state assignments for FeMoco are ˜ 5 cEuropean Synchrotron Radiation Facility, Boite Postale 220, 38043 Grenoble Cedex, discussed in the literature: (i) 6Fe(II)1Fe(III)Mo(IV); (ii) 4Fe(II) 6 7 France 3Fe(III)Mo(IV); and (iii) 2Fe(II)5Fe(III)Mo(IV). These can alter- dDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, New York nately be written in terms of total charge on the cluster when À 14853, USA sulfur and carbon are taken in their usual closed-shell form S2 † Electronic supplementary information (ESI) available: Further experimental and 4À 3À 1À and C , thus giving [MoFe7S9C] , [MoFe7S9C] and computational data and discussion. See DOI: 10.1039/c4sc00337c [MoFe S C]1+, respectively. For all of these oxidation state ‡ 7 9 These authors have contributed equally to the work presented in this ¼ manuscript. assignments, one can arrive at a total spin of S 3/2, consistent 8,9 § Present address: Centro Nacional de Pesquisa em Energia e Materiais, Brazilian with EPR spectroscopy. Interestingly, all of these assignments Synchrotron Light Laboratory – LNLS, CP 6192, 13084-971 Campinas, SP, Brazil. assume a closed-shell diamagnetic Mo(IV). { Present address: Howard Hughes Medical Institute and Division of Chemistry The Mo(IV) assignment in the FeMo cofactor is based on early and Chemical Engineering, California Institute of Technology, Pasadena CA Mo K-edge XAS studies and 95Mo ENDOR. As the molybdenum 91125, USA. 3096 | Chem. Sci.,2014,5,3096–3103 This journal is © The Royal Society of Chemistry 2014 View Article Online Edge Article Chemical Science atom can be probed directly by either of these methods, it serves as an important starting point for understanding the complex electronic structure of the cluster. The initial XAS studies were carried out in the 1970s and 1980s, before the structure of the cofactor was known. Based on Mo–S derived bond lengths from the EXAFS region, an oxidation state of either Mo(III) or Mo(IV) 10,11 was suggested. The absorption edge positions did not give a Fig. 1 Molecular structures of 3 (left), FeMoco (center) and an overlay clear indication of the Mo oxidation state, largely due to the of the two experimental X-ray structures (right). signicant core hole lifetime broadening at high energies. Later, 95Mo ENDOR experiments12–14 indicated a small Mo hyperne coupling in the protein and these data were interpreted as the In this work, we show that the detailed comparison of molybdenum most plausibly being a closed-shell Mo(IV)rather protein and model Mo-HERFD XAS data together with the than a S ¼ 3/2 Mo(III)orS ¼ 1/2 Mo(V). However, the authors results of density functional theory (DFT) calculations requires acknowledged that the spin coupling schemes utilized were a revised oxidation state assignment for molybdenum in the highly simplied and this assignment was not denitive.13 In resting state of FeMoco. Further, we demonstrate that the 15 3À 1988, a follow up Mo L-edge XAS study assigned the Mo atom in Mo(III) assignment holds for any of the proposed [MoFe7S9C] , 1À 1+ nitrogenase as Mo(IV), when taking the ENDOR data into account. [MoFe7S9C] or [MoFe7S9C] total charges on the cluster. However, a quantitative analysis of Mo L-edge data was prohibi- These results thus support Fe-based redox over this four elec- tive due to the proximity of the sulfur K-edge and the inadequacy tron series and suggest that a Mo(III) site could be mechanisti- of the available theoretical tools at that time. From this point on, cally relevant. the Mo(IV) oxidation state appeared to be set in the literature, despite the fact that the authors of both the XAS and ENDOR Results and discussion Creative Commons Attribution 3.0 Unported Licence. studies acknowledged the limitations of their methods. In the present study, we directly address the question of the Fig. 2 shows a comparison of the normalized Mo K-edge Mo oxidation state in FeMoco by using high-energy resolution HERFD-XAS data for compounds 1–4 and the intact MoFe uorescence detected X-ray absorption spectroscopy (HERFD- protein. As the only Mo present in the MoFe protein is associ- XAS) as a means to overcome standard XAS limitations. K-edge ated with FeMoco, the spectrum corresponds to a unique Mo HERFD-XAS detects uorescent photons resulting from 2p to 1s site. Compounds 1 and 2 clearly have the most intense pre- radiative decay as a function of the incident X-ray energy edges (at 20 003.6 and 20 004.0 eV) and the highest energy 16,17 scanned across an absorption edge. When this type of rising edges (at 20 009.9 and 20 011.5 eV), respectively, consis- measurement is carried out the resultant spectra have greatly tent with the tetrahedral local geometry and a Mo(V) oxidation This article is licensed under a – improved energy resolution (from 6 8 eV in standard XAS to state assignment. Complexes 3 and 4 have weaker pre-edge 3.5 eV in the present study). Hence, HERFD-XAS at the Mo K- intensities, consistent with the octahedral local site symmetry edge allows for more richly featured near-edge spectra than and lower energy rising edges (at 20 009.1 and 20 008.7 eV), Open Access Article.
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