All Publications


  • XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase. Journal of inorganic biochemistry Ohmer, C. J., Dasgupta, M., Patwardhan, A., Bogacz, I., Kaminsky, C., Doyle, M. D., Chen, P. Y., Keable, S. M., Makita, H., Simon, P. S., Massad, R., Fransson, T., Chatterjee, R., Bhowmick, A., Paley, D. W., Moriarty, N. W., Brewster, A. S., Gee, L. B., Alonso-Mori, R., Moss, F., Fuller, F. D., Batyuk, A., Sauter, N. K., Bergmann, U., Drennan, C. L., Yachandra, V. K., Yano, J., Kern, J. F., Ragsdale, S. W. 2022; 230: 111768

    Abstract

    Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances. While recent publications have proposed a radical-based catalytic mechanism as well as novel sulfonate-based binding modes of MCR for its native substrates, the structure of the active state of MCR, as well as a complete characterization of the reaction, remain elusive. Previous attempts to structurally characterize the active MCR-Ni(I) state have been unsuccessful due to oxidation of the redox- sensitive catalytic Ni center. Further, while many cryo structures of the inactive Ni(II)-enzyme in various substrates-bound forms have been published, no room temperature structures have been reported, and the structure and mechanism of MCR under physiologically relevant conditions is not known. In this study, we report the first room temperature structure of the MCRred1-silent Ni(II) form using an X-ray Free-Electron Laser (XFEL), with simultaneous X-ray Emission Spectroscopy (XES) and X-ray Diffraction (XRD) data collection. In celebration of the seminal contributions of inorganic chemist Dick Holm to our understanding of nickel-based catalysis, we are honored to announce our findings in this special issue dedicated to this remarkable pioneer of bioinorganic chemistry.

    View details for DOI 10.1016/j.jinorgbio.2022.111768

    View details for PubMedID 35202981

    View details for PubMedCentralID PMC8930625

  • Millisecond timescale reactions observed via X-ray spectroscopy in a 3D microfabricated fused silica mixer. Corrigendum. Journal of synchrotron radiation Huyke, D. A., Ramachandran, A., Ramirez-Neri, O., Guerrero-Cruz, J. A., Gee, L. B., Braun, A., Sokaras, D., Garcia-Estrada, B., Solomon, E. I., Hedman, B., Delgado-Jaime, M. U., DePonte, D. P., Kroll, T., Santiago, J. G. 2022; 29 (Pt 3): 930

    Abstract

    A figure in the article by Huyke et al. [(2021), J. Synchrotron Rad. 28, 1100-1113] is corrected.

    View details for DOI 10.1107/S1600577522002806

    View details for PubMedID 35511027

  • Carbon monoxide binding to alpha-R277H Mo-nitrogenase - Evidence for multiple pH-dependent species from IR-monitored photolysis. Journal of inorganic biochemistry Gee, L. B., Scott, A. D., Dapper, C. H., Newton, W. E., Cramer, S. P. 2022; 232: 111806

    Abstract

    The nitrogenase (N2ase) enzyme family is responsible for the conversion of dinitrogen into biologically accessible ammonia, a critical step in the global nitrogen cycle. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction, but it can also be reduced to hydrocarbons catalyzed by all three N2ases, namely the wild-type Mo enzyme and select variants and the V and Fe nitrogenases, both of which are orders of magnitude more effective. CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, we investigated the interaction of CO with the alpha-R277H variant of the Azotobacter vinelandii N2ase MoFe protein, in which the alpha-subunit 277Arg residue is replaced by His and results in production of only the S=3/2 EPR signal (denoted as hi(5)-CO). Fourier-transform infrared (FT-IR) spectroscopy was used to follow the photolysis of CO bound to the alpha-R277H variant under cryogenic conditions. Multiple EPR-silent species were observed with FT-IR spectroscopic signatures previously assigned to CO-inhibited forms of the alpha-H195Q and alpha-H195N N2ase variants. The distribution of these CO-inhibited forms varied dramatically with pH over the range of pH6.5 to pH8.5, indicating protonation/deprotonation involvement.

    View details for DOI 10.1016/j.jinorgbio.2022.111806

    View details for PubMedID 35439691

  • Nuclear Resonance Vibrational Spectroscopy: A Modern Tool to Pinpoint Site-Specific Cooperative Processes CRYSTALS Wang, H., Braun, A., Cramer, S. P., Gee, L. B., Yoda, Y. 2021; 11 (8)
  • NRVS and DFT of MitoNEET: Understanding the Special Vibrational Structure of a [2Fe-2S] Cluster with (Cys)(3)(His)(1) Ligation BIOCHEMISTRY Gee, L. B., Pelmenschikov, V., Mons, C., Mishra, N., Wang, H., Yoda, Y., Tamasaku, K., Golinelli-Cohen, M., Cramer, S. P. 2021; 60 (31): 2419-2424

    Abstract

    The human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron-sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer, and possibly Parkinson's disease. The geometric structure of mNT as a function of redox state and pH is critical for its function. In this study, we combine 57Fe nuclear resonance vibrational spectroscopy with density functional theory calculations to understand the novel properties of this important protein.

    View details for DOI 10.1021/acs.biochem.1c00252

    View details for Web of Science ID 000685204700008

    View details for PubMedID 34310123

    View details for PubMedCentralID PMC8672731

  • Millisecond timescale reactions observed via X-ray spectroscopy in a 3D microfabricated fused silica mixer. Journal of synchrotron radiation Huyke, D. A., Ramachandran, A., Ramirez-Neri, O., Guerrero-Cruz, J. A., Gee, L. B., Braun, A., Sokaras, D., Garcia-Estrada, B., Solomon, E. I., Hedman, B., Delgado-Jaime, M. U., DePonte, D. P., Kroll, T., Santiago, J. G. 2021; 28 (Pt 4): 1100-1113

    Abstract

    Determination of electronic structures during chemical reactions remains challenging in studies which involve reactions in the millisecond timescale, toxic chemicals, and/or anaerobic conditions. In this study, a three-dimensionally (3D) microfabricated microfluidic mixer platform that is compatible with time-resolved X-ray absorption and emission spectroscopy (XAS and XES, respectively) is presented. This platform, to initiate reactions and study their progression, mixes a high flow rate (0.50-1.5 ml min-1) sheath stream with a low-flow-rate (5-90 l min-1) sample stream within a monolithic fused silica chip. The chip geometry enables hydrodynamic focusing of the sample stream in3D and sample widths as small as 5 m. The chip is also connected to a polyimide capillary downstream to enable sample stream deceleration, expansion, and X-ray detection. In this capillary, sample widths of 50 m are demonstrated. Further, convection-diffusion-reaction models of the mixer are presented. The models are experimentally validated using confocal epifluorescence microscopy and XAS/XES measurements of a ferricyanide and ascorbic acid reaction. The models additionally enable prediction of the residence time and residence time uncertainty of reactive species as well as mixing times. Residence times (from initiation of mixing to the point of X-ray detection) during sample stream expansion as small as 2.1 ± 0.3 ms are also demonstrated. Importantly, an exploration of the mixer operational space reveals a theoretical minimum mixing time of 0.91 ms. The proposed platform is applicable to the determination of the electronic structure of conventionally inaccessible reaction intermediates.

    View details for DOI 10.1107/S1600577521003830

    View details for PubMedID 34212873

  • Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases. Proceedings of the National Academy of Sciences of the United States of America Iyer, S. R., Tidemand, K. D., Babicz, J. T., Jacobs, A. B., Gee, L. B., Haahr, L. T., Yoda, Y., Kurokuzu, M., Kitao, S., Saito, M., Seto, M., Christensen, H. E., Peters, G. H., Solomon, E. I. 2021; 118 (15)

    Abstract

    The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mossbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

    View details for DOI 10.1073/pnas.2022379118

    View details for PubMedID 33876764

  • Effect of 3d/4p Mixing on 1s2p Resonant Inelastic X-ray Scattering: Electronic Structure of Oxo-Bridged Iron Dimers. Journal of the American Chemical Society Kroll, T., Baker, M. L., Wilson, S. A., Lundberg, M., Juhin, A., Arrio, M., Yan, J. J., Gee, L. B., Braun, A., Weng, T., Sokaras, D., Hedman, B., Hodgson, K. O., Solomon, E. I. 2021

    Abstract

    1s2p resonant inelastic X-ray scattering (1s2p RIXS) has proven successful in the determination of the differential orbital covalency (DOC, the amount of metal vs ligand character in each d molecular orbital) of highly covalent centrosymmetric iron environments including heme models and enzymes. However, many reactive intermediates have noncentrosymmetric environments, e.g., the presence of strong metal-oxo bonds, which results in the mixing of metal 4p character into the 3d orbitals. This leads to significant intensity enhancement in the metal K-pre-edge and as shown here, the associated 1s2p RIXS features, which impact their insight into electronic structure. Binuclear oxo bridged high spin Fe(III) complexes are used to determine the effects of 4p mixing on 1s2p RIXS spectra. In addition to developing the analysis of 4p mixing on K-edge XAS and 1s2p RIXS data, this study explains the selective nature of the 4p mixing that also enhances the analysis of L-edge XAS intensity in terms of DOC. These 1s2p RIXS biferric model studies enable new structural insight from related data on peroxo bridged biferric enzyme intermediates. The dimeric nature of the oxo bridged Fe(III) complexes further results in ligand-to-ligand interactions between the Fe(III) sites and angle dependent features just above the pre-edge that reflect the superexchange pathway of the oxo bridge. Finally, we present a methodology that enables DOC to be obtained when L-edge XAS is inaccessible and only 1s2p RIXS experiments can be performed as in many metalloenzyme intermediates in solution.

    View details for DOI 10.1021/jacs.0c11193

    View details for PubMedID 33730507

  • Short-lived metal-centered excited state initiates iron-methionine photodissociation in ferrous cytochrome c. Nature communications Reinhard, M. E., Mara, M. W., Kroll, T., Lim, H., Hadt, R. G., Alonso-Mori, R., Chollet, M., Glownia, J. M., Nelson, S., Sokaras, D., Kunnus, K., Driel, T. B., Hartsock, R. W., Kjaer, K. S., Weninger, C., Biasin, E., Gee, L. B., Hodgson, K. O., Hedman, B., Bergmann, U., Solomon, E. I., Gaffney, K. J. 2021; 12 (1): 1086

    Abstract

    The dynamics of photodissociation and recombination in heme proteins represent an archetypical photochemical reaction widely used to understand the interplay between chemical dynamics and reaction environment. We report a study of the photodissociation mechanism for the Fe(II)-S bond between the heme iron and methionine sulfur of ferrous cytochrome c. This bond dissociation is an essential step in the conversion of cytochrome c from an electron transfer protein to a peroxidase enzyme. We use ultrafast X-ray solution scattering to follow the dynamics of Fe(II)-S bond dissociation and 1s3p (Kbeta) X-ray emission spectroscopy to follow the dynamics of the iron charge and spin multiplicity during bond dissociation. From these measurements, we conclude that the formation of a triplet metal-centered excited state with anti-bonding Fe(II)-S interactions triggers the bond dissociation and precedes the formation of the metastable Fe high-spin quintet state.

    View details for DOI 10.1038/s41467-021-21423-w

    View details for PubMedID 33597529

  • Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by 13C2H-ADT Labeling. Journal of the American Chemical Society Pelmenschikov, V., Birrell, J. A., Gee, L. B., Richers, C. P., Reijerse, E. J., Wang, H., Arragain, S., Mishra, N., Yoda, Y., Matsuura, H., Li, L., Tamasaku, K., Rauchfuss, T. B., Lubitz, W., Cramer, S. P. 2021

    Abstract

    [FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe]H subcluster. A -13C2H2- ADT labeling in the synthetic diiron precursor of [2Fe]H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), and NRVS was measured on samples poised in the catalytically crucial Hhyd state containing a terminal hydride at the distal Fe site. The 13C2H isotope effects were observed also in the Hhyd spectrum. DFT simulations of the spectra allowed identification of the 57Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H2 formation.

    View details for DOI 10.1021/jacs.1c02323

    View details for PubMedID 34043346

  • Nuclear Resonance Vibrational Spectroscopic Definition of the Fe(IV)2 Intermediate Q in Methane Monooxygenase and Its Reactivity. Journal of the American Chemical Society Jacobs, A. B., Banerjee, R., Deweese, D. E., Braun, A., Babicz, J. T., Gee, L. B., Sutherlin, K. D., Böttger, L. H., Yoda, Y., Saito, M., Kitao, S., Kobayashi, Y., Seto, M., Tamasaku, K., Lipscomb, J. D., Park, K., Solomon, E. I. 2021

    Abstract

    Methanotrophic bacteria utilize the nonheme diiron enzyme soluble methane monooxygenase (sMMO) to convert methane to methanol in the first step of their metabolic cycle under copper-limiting conditions. The structure of the sMMO Fe(IV)2 intermediate Q responsible for activating the inert C-H bond of methane (BDE = 104 kcal/mol) remains controversial, with recent studies suggesting both "open" and "closed" core geometries for its active site. In this study, we employ nuclear resonance vibrational spectroscopy (NRVS) to probe the geometric and electronic structure of intermediate Q at cryogenic temperatures. These data demonstrate that Q decays rapidly during the NRVS experiment. Combining data from several years of measurements, we derive the NRVS vibrational features of intermediate Q as well as its cryoreduced decay product. A library of 90 open and closed core models of intermediate Q is generated using density functional theory to analyze the NRVS data of Q and its cryoreduced product as well as prior spectroscopic data on Q. Our analysis reveals that a subset of closed core models reproduce these newly acquired NRVS data as well as prior data. The reaction coordinate with methane is also evaluated using both closed and open core models of Q. These studies show that the potent reactivity of Q toward methane resides in the "spectator oxo" of its Fe(IV)2O2 core, in contrast to nonheme mononuclear Fe(IV)═O enzyme intermediates that H atoms abstract from weaker C-H bonds.

    View details for DOI 10.1021/jacs.1c05436

    View details for PubMedID 34570980

  • High-Frequency Fe-H and Fe-H2 Modes in a trans-Fe(eta2-H2)(H) Complex: A Speed Record for Nuclear Resonance Vibrational Spectroscopy. Inorganic chemistry Chiang, M., Pelmenschikov, V., Gee, L. B., Liu, Y., Hsieh, C., Wang, H., Yoda, Y., Matsuura, H., Li, L., Cramer, S. P. 2020

    Abstract

    Nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) are complementary tools for studying the vibrational and geometric structures of specific isotopically labeled molecular systems. Here we apply NRVS and DFT to characterize the trans-[57Fe(eta2-H2)(H)(dppe)2][BPh4] [dppe = 1,2-bis(diphenylphosphino)ethane] complex. Heretofore, most NRVS observations have centered on the spectral region below 1000 cm-1, where the 57Fe signal is strongest. In this work, we show that state-of-the-art synchrotron facilities can extend the observable region to 2000 cm-1 and likely beyond, in measurements that require less than 1 day. The 57Fe-H stretch was revealed at 1915 cm-1, along with the asymmetric 57Fe-H2 stretch at 1774 cm-1. For a small fraction of the H2-dissociated product, the 57Fe-H stretch was detected at 1956 cm-1. The unique sensitivity to 57Fe motion and the isolated nature of the Fe-H/H2 stretching modes enabled NRVS to quantitatively analyze the sample composition.

    View details for DOI 10.1021/acs.inorgchem.0c03006

    View details for PubMedID 33356182

  • Valence-Dependent Electrical Conductivity in a 3D Tetrahydroxyquinone-Based Metal-Organic Framework. Journal of the American Chemical Society Chen, G., Gee, L. B., Xu, W., Zhu, Y., Lezama-Pacheco, J. S., Huang, Z., Li, Z., Babicz, J. T., Choudhury, S., Chang, T., Reed, E., Solomon, E. I., Bao, Z. 2020

    Abstract

    Electrically conductive metal-organic frameworks (cMOFs) have become a topic of intense interest in recent years because of their great potential in electrochemical energy storage, electrocatalysis, and sensing applications. Most of the cMOFs reported hitherto are 2D structures, and 3D cMOFs remain rare. Herein we report FeTHQ, a 3D cMOF synthesized from tetrahydroxy-1,4-quinone (THQ) and iron(II) sulfate salt. FeTHQ exhibited a conductivity of 3.3 ± 0.55 mS cm-1 at 300 K, which is high for 3D cMOFs. The conductivity of FeTHQ is valence-dependent. A higher conductivity was measured with the as-prepared FeTHQ than with the air-oxidized and sodium naphthalenide-reduced samples.

    View details for DOI 10.1021/jacs.0c09379

    View details for PubMedID 33315385

  • Single-ion magnetism in the extended solid-state: insights from X-ray absorption and emission spectroscopy CHEMICAL SCIENCE Huzan, M. S., Fix, M., Aramini, M., Bencok, P., Mosselmans, J. W., Hayama, S., Breitner, F. A., Gee, L. B., Titus, C. J., Arrio, M., Jesche, A., Baker, M. L. 2020; 11 (43): 11801–10

    View details for DOI 10.1039/d0sc03787g

    View details for Web of Science ID 000588192000010

  • Single-ion magnetism in the extended solid-state: insights from X-ray absorption and emission spectroscopy. Chemical science Huzan, M. S., Fix, M., Aramini, M., Bencok, P., Mosselmans, J. F., Hayama, S., Breitner, F. A., Gee, L. B., Titus, C. J., Arrio, M. A., Jesche, A., Baker, M. L. 2020; 11 (43): 11801-11810

    Abstract

    Large single-ion magnetic anisotropy is observed in lithium nitride doped with iron. The iron sites are two-coordinate, putting iron doped lithium nitride amongst a growing number of two coordinate transition metal single-ion magnets (SIMs). Uniquely, the relaxation times to magnetisation reversal are over two orders of magnitude longer in iron doped lithium nitride than other 3d-metal SIMs, and comparable with high-performance lanthanide-based SIMs. To understand the origin of these enhanced magnetic properties a detailed characterisation of electronic structure is presented. Access to dopant electronic structure calls for atomic specific techniques, hence a combination of detailed single-crystal X-ray absorption and emission spectroscopies are applied. Together K-edge, L2,3-edge and Kβ X-ray spectroscopies probe local geometry and electronic structure, identifying iron doped lithium nitride to be a prototype, solid-state SIM, clean of stoichiometric vacancies where Fe lattice sites are geometrically equivalent. Extended X-ray absorption fine structure and angular dependent single-crystal X-ray absorption near edge spectroscopy measurements determine FeI dopant ions to be linearly coordinated, occupying a D6h symmetry pocket. The dopant engages in strong 3dπ-bonding, resulting in an exceptionally short Fe-N bond length (1.873(7) Å) and rigorous linearity. It is proposed that this structure protects dopant sites from Renner-Teller vibronic coupling and pseudo Jahn-Teller distortions, enhancing magnetic properties with respect to molecular-based linear complexes. The Fe ligand field is quantified by L2,3-edge XAS from which the energy reduction of 3d z 2 due to strong 4s mixing is deduced. Quantification of magnetic anisotropy barriers in low concentration dopant sites is inhibited by many established methods, including far-infrared and neutron scattering. We deduce variable temperature L3-edge XAS can be applied to quantify the J = 7/2 magnetic anisotropy barrier, 34.80 meV (∼280 cm-1), that corresponds with Orbach relaxation via the first excited, MJ = ±5/2 doublet. The results demonstrate that dopant sites within solid-state host lattices could offer a viable alternative to rare-earth bulk magnets and high-performance SIMs, where the host matrix can be tailored to impose high symmetry and control lattice induced relaxation effects.

    View details for DOI 10.1039/d0sc03787g

    View details for PubMedID 34123206

    View details for PubMedCentralID PMC8162461

  • Vibrational characterization of a diiron bridging hydride complex - a model for hydrogen catalysis CHEMICAL SCIENCE Gee, L. B., Pelmenschikov, V., Wang, H., Mishra, N., Liu, Y., Yoda, Y., Tamasaku, K., Chiang, M., Cramer, S. P. 2020; 11 (21): 5487–93

    View details for DOI 10.1039/d0sc01290d

    View details for Web of Science ID 000538158300010

  • Vibrational characterization of a diiron bridging hydride complex - a model for hydrogen catalysis. Chemical science Gee, L. B., Pelmenschikov, V., Wang, H., Mishra, N., Liu, Y. C., Yoda, Y., Tamasaku, K., Chiang, M. H., Cramer, S. P. 2020; 11 (21): 5487-5493

    Abstract

    A diiron complex containing a bridging hydride and a protonated terminal thiolate of the form [(μ,κ2-bdtH)(μ-PPh2)(μ-H)Fe2(CO)5]+ has been investigated through 57Fe nuclear resonance vibrational spectroscopy (NRVS) and interpreted using density functional theory (DFT) calculations. We report the Fe-μH-Fe wagging mode, and indications for Fe-μD stretching vibrations in the D-isotopologue, observed by 57Fe-NRVS. Our combined approach demonstrates an asymmetric sharing of the hydride between the two iron sites that yields two nondegenerate Fe-μH/D stretching vibrations. The studied complex provides an important model relevant to biological hydrogen catalysis intermediates. The complex mimics proposals for the binuclear metal sites in [FeFe] and [NiFe] hydrogenases. It is also an appealing prototype for the 'Janus intermediate' of nitrogenase, which has been proposed to contain two bridging Fe-H-Fe hydrides and two protonated sulfurs at the FeMo-cofactor. The significance of observing indirect effects of the bridging hydride, as well as obstacles in its direct observation, is discussed in the context of biological hydrogen intermediates.

    View details for DOI 10.1039/d0sc01290d

    View details for PubMedID 34094075

    View details for PubMedCentralID PMC8159291

  • Tuning the Geometric and Electronic Structure of Synthetic High-Valent Heme Iron(IV)-Oxo Models in the Presence of a Lewis Acid and Various Axial Ligands. Journal of the American Chemical Society Ehudin, M. A., Gee, L. B., Sabuncu, S. n., Braun, A. n., Moënne-Loccoz, P. n., Hedman, B. n., Hodgson, K. O., Solomon, E. I., Karlin, K. D. 2019; 141 (14): 5942–60

    Abstract

    High-valent ferryl species (e.g., (Por)FeIV═O, Cmpd-II) are observed or proposed key oxidizing intermediates in the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in biological respiration and oxidative metabolism. Herein, various axially ligated iron(IV)-oxo complexes were prepared to examine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimidazole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Characterization was carried out via UV-vis, electron paramagnetic resonance (EPR), 57Fe Mössbauer, Fe X-ray absorption (XAS), and 54/57Fe resonance Raman (rR) spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric structures of these heme iron(IV)-oxo species. Mössbauer studies confirmed that the axially ligated derivatives were iron(IV) and six-coordinate complexes. XAS and 54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the protic Lewis acid, 2,6-lutidinium triflate (LutH+), with (or without) DCHIm. Mössbauer, rR, and XAS spectroscopic data indicated the formation of molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl species with or without Lewis acid vary drastically; addition of LutH+ to F8Cmpd-II (F8 = tetrakis(2,6-difluorophenyl)porphyrinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can increase the reactivity of ferryl species.

    View details for PubMedID 30860832

  • High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Pelmenschikov, V., Gee, L. B., Wang, H., MacLeod, K., McWilliams, S. F., Skubi, K. L., Cramer, S. P., Holland, P. L. 2018; 57 (30): 9367–71

    Abstract

    High-spin iron species with bridging hydrides have been detected in species trapped during nitrogenase catalysis, but there are few general methods of evaluating Fe-H bonds in high-spin multinuclear iron systems. An 57 Fe nuclear resonance vibrational spectroscopy (NRVS) study on an Fe(μ-H)2 Fe model complex reveals Fe-H stretching vibrations for bridging hydrides at frequencies greater than 1200 cm-1 . These isotope-sensitive vibrational bands are not evident in infrared (IR) spectra, showing the power of NRVS for identifying hydrides in this high-spin iron system. Complementary density functional theory (DFT) calculations elucidate the normal modes of the rhomboidal iron hydride core.

    View details for PubMedID 29847703

  • Sterically Stabilized Terminal Hydride of a Diiron Dithiolate INORGANIC CHEMISTRY Carlson, M. R., Gray, D. L., Richers, C. P., Wang, W., Zhao, P., Rauchfuss, T. B., Pelmenschikov, V., Pham, C. C., Gee, L. B., Wang, H., Cramer, S. P. 2018; 57 (4): 1988–2001

    Abstract

    The kinetically robust hydride [t-HFe2(Me2pdt)(CO)2(dppv)2]+ ([t-H1]+) (Me2pdt2- = Me2C(CH2S-)2; dppv = cis-1,2-C2H2(PPh2)2) and related derivatives were prepared with 57Fe enrichment for characterization by NMR, FT-IR, and NRVS. The experimental results were rationalized using DFT molecular modeling and spectral simulations. The spectroscopic analysis was aimed at supporting assignments of Fe-H vibrational spectra as they relate to recent measurements on [FeFe]-hydrogenase enzymes. The combination of bulky Me2pdt2- and dppv ligands stabilizes the terminal hydride with respect to its isomerization to the 5-16 kcal/mol more stable bridging hydride ([μ-H1]+) with t1/2(313.3 K) = 19.3 min. In agreement with the nOe experiments, the calculations predict that one methyl group in [t-H1]+ interacts with the hydride with a computed CH···HFe distance of 1.7 Å. Although [t-H571]+ exhibits multiple NRVS features in the 720-800 cm-1 region containing the bending Fe-H modes, the deuterated [t-D571]+ sample exhibits a unique Fe-D/CO band at ∼600 cm-1. In contrast, the NRVS spectra for [μ-H571]+ exhibit weaker bands near 670-700 cm-1 produced by the Fe-H-Fe wagging modes coupled to Me2pdt2- and dppv motions.

    View details for DOI 10.1021/acs.inorgchem.7b02903

    View details for Web of Science ID 000426014800033

    View details for PubMedID 29384371

    View details for PubMedCentralID PMC5821139

  • NRVS for Fe in Biology: Experiment and Basic Interpretation. Methods in enzymology Gee, L. B., Wang, H., Cramer, S. P. 2018; 599: 409–25

    Abstract

    For over 20 years, nuclear resonance vibrational spectroscopy (NRVS) has been used to study vibrational dynamics of iron-containing materials. With the only selection rule being iron motion, 57Fe NRVS has become an excellent tool to study iron-containing enzymes. Over the past decade, considerable progress has been made in the study of complex metalloenzymes using NRVS. Iron cofactors in heme-containing globins; [2Fe2S], [3Fe4S], [4Fe4S] proteins; the [NiFe] and [FeFe] hydrogenases; and nitrogenases have been explored in a fashion not possible through traditional vibrational spectroscopy. In this chapter, we discuss the basics of NRVS, a strategy to perform NRVS, and a discussion of the application of NRVS on rubredoxin and [FeFe] hydrogenase.

    View details for DOI 10.1016/bs.mie.2017.11.002

    View details for PubMedID 29746248

  • Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Reijerse, E. J., Pham, C. C., Pelmenschikov, V., Gilbert-Wilson, R., Adamska-Venkatesh, A., Siebel, J. F., Gee, L. B., Yoda, Y., Tamasaku, K., Lubitz, W., Rauchfuss, T. B., Cramer, S. P. 2017; 139 (12): 4306–9

    Abstract

    [FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN- and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.

    View details for DOI 10.1021/jacs.7b00686

    View details for Web of Science ID 000398247100018

    View details for PubMedID 28291336

    View details for PubMedCentralID PMC5545132

  • Is trehalose an effective quenching agent of Azotobacter vinelandii Mo-nitrogenase turnover? INORGANICA CHIMICA ACTA Gee, L. B., Scott, A. D., Dapper, C. H., Newton, W. E., Cramer, S. P. 2016; 453: 74–77
  • Synchrotron-based Nickel Mossbauer Spectroscopy INORGANIC CHEMISTRY Gee, L. B., Lin, C., Jenney, F. E., Adams, M. W., Yoda, Y., Masuda, R., Saito, M., Kobayashi, Y., Tamasaku, K., Lerche, M., Seto, M., Riordan, C. G., Ploskonka, A., Power, P. P., Cramer, S. P., Lauterbach, L. 2016; 55 (14): 6866–72

    Abstract

    We used a novel experimental setup to conduct the first synchrotron-based (61)Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. (61)NiCr2O4 was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional (61)Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, (61)Ni{N(SiMe3)Dipp}2, where Dipp = C6H3-2,6-(i)Pr2, was chosen as a sample with an "extreme" geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based (61)Ni Mössbauer spectroscopy, we examined the spectra of (61)Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et4N]2[(61)Ni(SPh)4] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based (61)Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources.

    View details for DOI 10.1021/acs.inorgchem.5b03004

    View details for Web of Science ID 000380181400011

    View details for PubMedID 27387959

  • Characterization of the [3Fe-4S](0/1+) cluster from the D14C variant of Pyrococcus furiosus ferredoxin via combined NRVS and DFT analyses DALTON TRANSACTIONS Lauterbach, L., Gee, L. B., Pelmenschikov, V., Jenney, F. E., Kamali, S., Yoda, Y., Adams, M. W., Cramer, S. P. 2016; 45 (17): 7215–19

    Abstract

    The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe-4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe-4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe-S clusters.

    View details for DOI 10.1039/c5dt04760a

    View details for Web of Science ID 000375001000005

    View details for PubMedID 27063792

    View details for PubMedCentralID PMC4940129

  • Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration JOURNAL OF INORGANIC BIOCHEMISTRY Maiuri, M., Delfino, I., Cerullo, G., Manzoni, C., Pelmenschikov, V., Guo, Y., Wang, H., Gee, L. B., Dapper, C. H., Newton, W. E., Cramer, S. P. 2015; 153: 128–35

    Abstract

    We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226cm(-1) band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group.

    View details for DOI 10.1016/j.jinorgbio.2015.07.005

    View details for Web of Science ID 000367563200013

    View details for PubMedID 26343576

    View details for PubMedCentralID PMC4917305

  • Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy NATURE COMMUNICATIONS Ogata, H., Kraemer, T., Wang, H., Schilter, D., Pelmenschikov, V., van Gastel, M., Neese, F., Rauchfuss, T. B., Gee, L. B., Scott, A. D., Yoda, Y., Tanaka, Y., Lubitz, W., Cramer, S. P. 2015; 6: 7890

    Abstract

    The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the (57)Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique 'wagging' mode involving H(-) motion perpendicular to the Ni(μ-H)(57)Fe plane was studied using (57)Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)(57)Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe-CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)(57)Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)(57)Fe(CO)3](+) and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H(-) binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe-H moieties in other important natural and synthetic catalysts.

    View details for DOI 10.1038/ncomms8890

    View details for Web of Science ID 000360344600002

    View details for PubMedID 26259066

    View details for PubMedCentralID PMC4531378

  • Docking and Migration of Carbon Monoxide in Nitrogenase: The Case for Gated Pockets from Infrared Spectroscopy and Molecular Dynamics BIOCHEMISTRY Gee, L. B., Leontyev, I., Stuchebrukhov, A., Scott, A. D., Pelmenschikov, V., Cramer, S. P. 2015; 54 (21): 3314–19

    Abstract

    Evidence of a CO docking site near the FeMo cofactor in nitrogenase has been obtained by Fourier transform infrared spectroscopy-monitored low-temperature photolysis. We investigated the possible migration paths for CO from this docking site using molecular dynamics calculations. The simulations support the notion of a gas channel with multiple internal pockets from the active site to the protein exterior. Travel between pockets is gated by the motion of protein residues. Implications for the mechanism of nitrogenase reactions with CO and N2 are discussed.

    View details for DOI 10.1021/acs.biochem.5b00216

    View details for Web of Science ID 000355779600007

    View details for PubMedID 25919807

    View details for PubMedCentralID PMC4522414

  • Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O-2-tolerant NAD(+)-reducing [NiFe] hydrogenase CHEMICAL SCIENCE Lauterbach, L., Wang, H., Horch, M., Gee, L. B., Yoda, Y., Tanaka, Y., Zebger, I., Lenz, O., Cramer, S. P. 2015; 6 (2): 1055–60

    Abstract

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

    View details for DOI 10.1039/c4sc02982h

    View details for Web of Science ID 000348147100024

    View details for PubMedID 25678951

    View details for PubMedCentralID PMC4321745

  • Synthesis and vibrational spectroscopy of Fe-57-labeled models of [NiFe] hydrogenase: first direct observation of a nickel-iron interaction CHEMICAL COMMUNICATIONS Schilter, D., Pelmenschikov, V., Wang, H., Meier, F., Gee, L. B., Yoda, Y., Kaupp, M., Rauchfuss, T. B., Cramer, S. P. 2014; 50 (88): 13469–72

    Abstract

    A new route to iron carbonyls has enabled synthesis of (57)Fe-labeled [NiFe] hydrogenase mimic (OC)3(57)Fe(pdt)Ni(dppe). Its study by nuclear resonance vibrational spectroscopy revealed Ni-(57)Fe vibrations, as confirmed by calculations. The modes are absent for [(OC)3(57)Fe(pdt)Ni(dppe)](+), which lacks Ni-(57)Fe bonding, underscoring the utility of the analyses in identifying metal-metal interactions.

    View details for DOI 10.1039/c4cc04572f

    View details for Web of Science ID 000343965300010

    View details for PubMedID 25237680

    View details for PubMedCentralID PMC4191989

  • Differences in peripheral endocannabinoid modulation of scratching behavior in facial vs. spinally-innervated skin NEUROPHARMACOLOGY Spradley, J., Davoodi, A., Gee, L., Carstens, M., Carstens, E. 2012; 63 (4): 743–49

    Abstract

    Cannabinoids suppress nocifensive behaviors in rodents. We presently investigated peripheral endocannabinoid modulation of itch- and pain-related behaviors elicited from facial vs. spinally-innervated skin of rats. Intradermal (id) injection of the pruritogen serotonin (5-HT) elicited significantly more hindlimb scratch bouts, and longer cumulative time scratching, when injected in the rostral back compared to the cheek. Pretreatment of skin with inhibitors of degrading enzymes for the endocannabinoids anandamide (URB597) or 2-arachidonoylglycerol (JZL184) significantly reduced scratching elicited by 5-HT in the rostral back. These effects were prevented by co-treatment with antagonists of the CB₁ (AM251) or CB₂ receptor (AM630), implicating both receptor subtypes in endocannabinoid suppression of scratching in spinally-innervated skin. Conversely, pretreatment with either enzyme inhibitor, or with AM630 alone, increased the number of scratch bouts elicited by id 5-HT injection in the cheek. Moreover, pretreatment with JZL184 also significantly increased pain-related forelimb wipes directed to the cheek following id injection of the algogen, allyl isothiocyanate (AITC; mustard oil). Thus, peripheral endocannabinoids have opposite effects on itch-related scratching behaviors in trigeminally- vs. spinally-innervated skin. These results suggest that increasing peripheral endocannabinoid levels represents a promising therapeutic approach to treat itch arising from the lower body, but caution that such treatment may not relieve, and may even exacerbate, itch and pain arising from trigeminally-innervated skin of the face or scalp.

    View details for DOI 10.1016/j.neuropharm.2012.05.032

    View details for Web of Science ID 000306637700026

    View details for PubMedID 22683515

    View details for PubMedCentralID PMC3394407