Lessons from X-Ray Spectroscopic Studies

In search of Nature-inspired catalytic system: lessons from X-ray spectroscopic studies

Joanna K. Kowalska

Kraków, 03 November 2016 The Institute of Nuclear Physics Polish Academy of Sciences Motivation: our needs are increasing…

United Nations, Department of Economic and Social Affairs, Population Division (2013). World Population Prospects: The Revision, Highlights and Advance Tables. Working Paper No. ESA/P/WP.228. One of the sources: nitrogen fixation

N2 → 2 NH3 Bioavailable nitrogen source needed: - amino acids  proteins - nucleic acids  DNA, RNA

 Medicaments &  Dyes, Resins  Fertilizers  Gums

Nitrogen source

Industrial process Biological fixation • Haber – Bosch • Nitrogenase enzyme • 300 – 400 ºC • Found in diazotrophs • 90 – 300 atm • Ambient conditions Nitrogen source

Industrial process Biological fixation • Haber – Bosch • Nitrogenase enzyme • 300 – 400 ºC • Found in diazotrophs • 90 – 300 atm • Ambient conditions uses complex Fe surfaces Fe-Mo cofactor of to effect this conversion nitrogenase enzyme

What/How Nature does it better?

The need to design a catalyst that will compete with both systems.

“If you want to understand function, study structure.” Francis Crick Nitrogenase

+ - N2 + 8H + 16MgATP + 8e → 2 NH3 + H2 + 16MgADP + 16Pi

- 2 metalocluster:

• P-cluster [Fe8S7] • MoFe7S9 cofactor – active site

Seefeldt, Hoffman, Dean, Ann. Rev. Biochem. (2009) 78:701-22 Towards understanding the mechanism of N2 reduction…

An understanding of the molecular level mechanism We are here requires a detailed understanding of the electronic structure of the cofactor (at each stage)

Requires knowledge of:

• Atomic composition • Charge distribution • Oxidation state distribution • Magnetic coupling

Elemental specific technique: X-ray spectroscopy X-ray Source  Synchrotron

European Synchrotron Radiation Facility, Grenoble FR National French Synchrotron, SOLEIL,, Gif-sur-Yvette, Paris, FR Electron Why SR?

 High Brilliance  Tunable energy (whole spectrum)  Small focus Picture credits:  Polarization Dr. Iwona Sputowska, IFJ PAN Synchrotron radiation The Brief History of X…

 1992: Rees et al. published the first structure of FeMoco

 2002: Einsle et al. crystal structure revealed electron density (C4-, N3- or O2- …?)

 Rees et al. favor N  2003-2005: EPR (Hoffman): not a nitrogen

 DFT (Dance, Noodleman): initially favor X=N  2007: DFT, ENDOR, ESEEM (Hoffman, Noodleman) X not C or N…

 2008: QM/MM (Cao): C-centered MoFe

 2008: NRVS/EXAFS (Cramer): consistent with the presence of a light atom but cannot identify it  … The answer came from X-ray spectroscopy… Fe XES X-ray Emission Spectroscopy (XES) - K - Kβ (mainline) - Valenceα -to-core

Ligands attached to Fe: S, X…

J.K. Kowalska, F.A. Lima, C.J. Pollock, J.A. Rees, S. DeBeer Isr J Chem (2016) 56: 803-815 K.M. Lancaster, M. Roemelt, P. Ettenhuber, Y. Hu, M.W. Ribbe, F. Neese, U. Bergmann S. DeBeer Science (2011) 334: 974

Fe7MoSFe7MoS9X – Fe9X 8–C 7Fe 8C7 … supported by Density Functional Theory (DFT) calculations

Carbon in the middle!

C:2s  Fe:1s

N:2s  Fe:1s O:2s  Fe:1s Complete atomic composition

MoFe7S9C

1992 2002 2011

K.M. Lancaster, M. Roemelt, P. Ettenhuber, Y. Hu, M.W. Ribbe, F. Neese, U. Bergmann S. DeBeer Science (2011) 334: 974 J. Kowalska, S. DeBeer Biol Biochem Acta MCR (2015) 1853: 1406-1415 • Atomic composition √ • Charge distribution ? • Oxidation state distribution ? • Magnetic coupling ?

Oxidation state distribution MoFe7S9C

What has been known:  Total spin = 3/2 (ground state)

 proposals of oxidation states:

• [MoFe7S9C]1+ : 5Fe(III) : 2Fe(II) : Mo(IV) • [MoFe7S9C]1- : 3Fe(III) : 4Fe(II) : Mo(IV) • [MoFe7S9C]3- : 1Fe(III) : 6Fe(II) : Mo(IV)

But why Mo(IV) ? The brief history of Mo oxidation state assignment

1976: Cramer & Hodgson propose Mo(V) based on initial Mo K-edge X-ray spectroscopic studies

1978-1985: Cramer, Conradson & Hodgson propose a Mo(III) or Mo(IV) assignment based on Mo-S bond distances from Extended X-ray Absorption Fine Structure studies

1986: Hoffman & Orme-Johnson report 95-Mo ENDOR (EPR + NMR); indicating a Mo(IV)

1989: Hedman & Hodgson report Mo L-edge X-ray Absorption data consistent with Mo(III) or Mo(IV)

For ~25 years Mo(IV) generally assumed…

2014: As there’s no clear answer - need to revisit this assignment… How to determine the oxidation state of an atom?

K-edge X-ray Absorption Spectroscopy (XAS)

Energy edge corresponds to the ionization energy  indicator of the oxidation state Pre-edge – position and number of the peaks are sensitive to the oxidation state, also symmetry, covalency…

J.K. Kowalska, F.A. Lima, C.J. Pollock, J.A. Rees, S. DeBeer Isr J Chem (2016) 56: 803-815 What we know more, what can we improve…

 We have more information: complete atomic composition

 The possibility of new types of detection

Standard XAS vs. high-resolution XAS detection XAS is measured as proportional to fluorescence Total fluorescence detection Resultant XAS spectrum

Kα: 2p1s

Scatter Kβ: 2p1s

Energy [eV] Energy [eV]

K. Hämäläinen, D.P. Siddons, J.B. Hastings, L.E. Berman Phys. Rev. Lett. (1991) 67:2850-2853 Adapted from Serena DeBeer (MPI CEC) Standard XAS vs. high-resolution XAS detection Solid State Detectors can repress the “scatter” background Partial fluorescence detection Resultant XAS spectrum

Kα: 2p1s

Scatter Kβ: 2p1s

Energy [eV] Energy [eV]

K. Hämäläinen, D.P. Siddons, J.B. Hastings, L.E. Berman Phys. Rev. Lett. (1991) 67:2850-2853 Adapted from Serena DeBeer (MPI CEC) Standard XAS vs. high-resolution fluorescence detected (HERFD) XAS High-energy resolution fluorescence detection XAS uses a ~2 eV slide through the Kα line Resolution dominated by 2p rather than 1s core hole.

HERFD detection Resultant XAS spectrum

Kα: 2p1s

HERFD Scatter PFY Kβ: 2p1s

Energy [eV] Energy [eV]

K. Hämäläinen, D.P. Siddons, J.B. Hastings, L.E. Berman Phys. Rev. Lett. (1991) 67:2850-2853 Adapted from Serena DeBeer (MPI CEC) Having access to high resolution setups & model complexes Mo K-edge high- energy resolution fluorescence detected XAS Mo(III)Fe(III,III,II) Mo(V)Fe(III) Mo(V)Fe(III) [Mo(III)Fe(III,III,II)]2

Mo K-edge data of FeMoco appear to FeMoco lowest energy.

Does NOT support a Mo(IV) assignment; supports Mo(III)

To our knowledge, this is the first experimental evidence for a Mo(III) site in biology.

R.Bjornsson, F.A.Lima, T. Spatzel, T.Weyhermueller, P.Glatzel, E.Bill, O.Einsle, F.Neese, S.DeBeer Chem. Sci. (2014) 5: 3096-3103 R. Bjornsson, F. Neese, R.R. Schrock, O. Einsle, S. DeBeer J Biol Inorg Chem (2015) 20: 447-460 J. Kowalska, S. DeBeer Biol Biochem Acta MCR (2015) 1853: 1406-1415 Experiment vs Theory: Mo HERFD XAS

Mo(III)Fe(III,III,II) Mo(V)Fe(III) Mo(V)Fe(III) [Mo(III)Fe(III,III,II)]2

Experiment Theory Time-dependent Density Functional Theory (TDDFT) 1 2 1 3 2 4 3 FeMoco 4 FeMoco

Both energy and intensity distributions are well-reproduced by theory.

- Complete atomic composition Fe7MoS9C - Mo(III)

- From EPR Stotal=3/2

This assignment of Mo (III) requires consideration of different oxidation state distributions:

• [MoFe7S9C]1+ : 5Fe(III) : 2Fe(II) : Mo(IV)  [MoFe7S9C]1+ : 6Fe(III) : 1Fe(II) : Mo(III) • [MoFe7S9C]1- : 3Fe(III) : 4Fe(II) : Mo(IV)  [MoFe7S9C]1- : 4Fe(III) : 3Fe(II) : Mo(III) • [MoFe7S9C]3- : 1Fe(III) : 6Fe(II) : Mo(IV)  [MoFe7S9C]3- : 2Fe(III) : 5Fe(II) : Mo(III)

Idea: Fe K-edge HERFD XAS Fe K-edge HERFD XAS

1.2 FeMoco MoFe 1.0

0.8 P- cluster II,III II 0.6 7 x Fe + 8 x Fe Fe8:S7 0.4 7 x FeII,III

Norm. intensity [a.u.] intensity Norm. 0.2

0.0 7110 7115 7120 7125 7130 Too many irons! Energy [eV] Adding Mo-Fe-S model complexes

C: Mo(V)Fe(III) D: Mo(V)Fe(III) G: Mo(III)Fe(III, III, II) F: [Mo(III)Fe(III, III, II)]2

D 0.15 F G

0.10 1.2 C D 1.0 F 0.05 G Norm. intensity [a.u.] intensity Norm.

0.8 Experiment 0.00 0.6 7110 7111 7112 7113 7114 7115 Energy [eV] Not bad!  0.4 C D 0.2 2.0 F Norm. intensity [a.u.] intensity Norm. G 1.6 0.0 7110 7115 7120 7125 7130 1.2 Energy [eV] 0.8

0.4 Calculated intensity

0.0

7020 7021 7022 7023 7024 7025 Calculations Energy [eV] Adding FeMoco

1- 1.2 FeMoco C 1.0 D F G 0.8

0.6

0.4

Norm. intensity [a.u.] intensity Norm. 0.2

0.0 7110 7115 7120 7125 7130 Compound G Holm cubane Energy [eV]

Mo(III)-Fe(III)2Fe(II)

Mo_Holm 1.2 MoFe FeMoco 1.0

0.8 0.6 Influence of central C 0.4

Norm. intensity [a.u.] intensity Norm. 0.2

0.0 Need more… 7110 7115 7120 7125 7130 7135 Energy [eV] FeMoco “More”… L3 1. L-edge X-ray Absorption Spectroscopy L3 - 2p  3d  L edge - Spin-orbit coupling:  L & L edge 2 3 L2 1 3 2𝑝𝑝 ⁄2 2𝑝𝑝 ⁄2 3d 3p L3 L2 - Larger lifetime  smaller broadening - Reality: multiplets 2p3/2 2p

2p1/2

Metal Ligand K-edge Pre-edge

Edge Energy [eV] Natural widths [eV] K 7112 1.25

L2 719.9 0.37

L3 706.8 0.36 2. X-ray Magnetic Circular Dichroism (XMCD)

Adding polarized light to L-edge XAS mf-mi=mph Convention: CL excites ↑

Selectively excitement of spin-up & spin-down electrons

J.K. Kowalska, B.Nayyar, J.A. Rees, S.C. Lee, F. Meyer, T. Weyhermüller, E. Otero, S. DeBeer (2016) submitted Lessons from X-ray spectroscopic studies:

FeMoco: - Carbon in the middle - Mo(III) assignment

Future:

- Towards assignment of the iron oxidation state distribution in FeMoco - Magnetic coupling Mo/Fe

Open questions:

- Role of the central carbon - Role of the heterometal in reactivity: FeMoco, FeVco nitrogenases

- Many more towards the mechanistm of N2 reduction Many, many thanks to:

. Serena DeBeer & the group (MPI CEC) . Ragnar Bjornsson (Univ. Iceland) . Alexander Nyrow (MPI CEC/Volkswagen) . Mario U. Delgado–Jaime (Univ. Utrecht) . Frederico A. Lima (European XFEL) . Pieter Glatzel (ESRF) . Edwige Otero (SOLEIL)

People who cooked the samples: . Oliver Einsle (Univ. Freiburg) . Thomas Weyhermüller (MPI CEC) . Sonny Lee (Univ. Waterloo) . Franc Meyer (Univ. Göttingen) . Julian Rees (Univ. Washington)

Funding $$$:

Thank you for your attention!!!