Boards, Advisory Committees, Professional Organizations

  • Member, Women Chemists Committee (2011 - 2015)
  • Member, American Chemical Society (ACS) (2010 - Present)

Professional Education

  • Bachelor of Science, Allegheny College (2009)
  • Doctor of Philosophy, Carnegie Mellon University (2015)

Stanford Advisors

All Publications

  • Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation. Proceedings of the National Academy of Sciences of the United States of America Appel, M. J., Meier, K. K., Lafrance-Vanasse, J., Lim, H., Tsai, C., Hedman, B., Hodgson, K. O., Tainer, J. A., Solomon, E. I., Bertozzi, C. R. 2019


    The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Calpha-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

    View details for PubMedID 30824597

  • Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars CHEMICAL REVIEWS Meier, K. K., Jones, S. M., Kaper, T., Hansson, H., Koetsier, M. J., Karkehabadi, S., Solomon, E. I., Sandgren, M., Kelemen, B. 2018; 118 (5): 2593–2635


    Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars. One of the major challenges facing large-scale industrial applications utilizing natural carbohydrate polymers is rooted in the fact that naturally occurring forms of starch, cellulose, and chitin can have tightly packed organizations of polymer chains with low hydration levels, giving rise to crystalline structures that are highly recalcitrant to enzymatic degradation. The topic of this review is oxidative cleavage of carbohydrate polymers by lytic polysaccharide mono-oxygenases (LPMOs). LPMOs are copper-dependent enzymes (EC that, with glycoside hydrolases, participate in the degradation of recalcitrant carbohydrate polymers. Their activity and structural underpinnings provide insights into biological mechanisms of polysaccharide degradation.

    View details for PubMedID 29155571

    View details for PubMedCentralID PMC5982588

  • High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain JOURNAL OF BIOLOGICAL CHEMISTRY Hansson, H., Karkehabadi, S., Mikkelsen, N., Douglas, N. R., Kim, S., Lam, A., Kaper, T., Kelemen, B., Meier, K. K., Jones, S. M., Solomon, E. I., Sandgren, M. 2017; 292 (46): 19099–109


    For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.

    View details for PubMedID 28900033

    View details for PubMedCentralID PMC5704490

  • New insight into the reaction mechanism of the formylglycine generating enzyme: A spectroscopic perspective Meier, K., Appel, M., Solomon, E. AMER CHEMICAL SOC. 2017
  • Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mossbauer and Computational Studies INORGANIC CHEMISTRY Meier, K. K., Rogers, M. S., Kovaleva, E. G., Lipscomb, J. D., Bominaar, E. L., Munck, E. 2016; 55 (12): 5862-5870


    The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

    View details for DOI 10.1021/acs.inorgchem.6b00148

    View details for Web of Science ID 000378369900021

    View details for PubMedID 27275865

  • Trapping a Highly Reactive Nonheme Iron Intermediate That Oxygenates Strong C-H Bonds with Stereoretention. Journal of the American Chemical Society Serrano-Plana, J., Oloo, W. N., Acosta-Rueda, L., Meier, K. K., Verdejo, B., García-España, E., Basallote, M. G., Münck, E., Que, L., Company, A., Costas, M. 2015; 137 (50): 15833-15842


    An unprecedentedly reactive iron species (2) has been generated by reaction of excess peracetic acid with a mononuclear iron complex [Fe(II)(CF3SO3)2(PyNMe3)] (1) at cryogenic temperatures, and characterized spectroscopically. Compound 2 is kinetically competent for breaking strong C-H bonds of alkanes (BDE ≈ 100 kcal·mol(-1)) through a hydrogen-atom transfer mechanism, and the transformations proceed with stereoretention and regioselectively, responding to bond strength, as well as to steric and polar effects. Bimolecular reaction rates are at least an order of magnitude faster than those of the most reactive synthetic high-valent nonheme oxoiron species described to date. EPR studies in tandem with kinetic analysis show that the 490 nm chromophore of 2 is associated with two S = 1/2 species in rapid equilibrium. The minor component 2a (∼5% iron) has g-values at 2.20, 2.19, and 1.99 characteristic of a low-spin iron(III) center, and it is assigned as [Fe(III)(OOAc)(PyNMe3)](2+), also by comparison with the EPR parameters of the structurally characterized hydroxamate analogue [Fe(III)(tBuCON(H)O)(PyNMe3)](2+) (4). The major component 2b (∼40% iron, g-values = 2.07, 2.01, 1.95) has unusual EPR parameters, and it is proposed to be [Fe(V)(O)(OAc)(PyNMe3)](2+), where the O-O bond in 2a has been broken. Consistent with this assignment, 2b undergoes exchange of its acetate ligand with CD3CO2D and very rapidly reacts with olefins to produce the corresponding cis-1,2-hydroxoacetate product. Therefore, this work constitutes the first example where a synthetic nonheme iron species responsible for stereospecific and site selective C-H hydroxylation is spectroscopically trapped, and its catalytic reactivity against C-H bonds can be directly interrogated by kinetic methods. The accumulated evidence indicates that 2 consists mainly of an extraordinarily reactive [Fe(V)(O)(OAc)(PyNMe3)](2+) (2b) species capable of hydroxylating unactivated alkyl C-H bonds with stereoretention in a rapid and site-selective manner, and that exists in fast equilibrium with its [Fe(III)(OOAc)(PyNMe3)](2+) precursor.

    View details for DOI 10.1021/jacs.5b09904

    View details for PubMedID 26599834

  • Upside Down! Crystallographic and Spectroscopic Characterization of an [Fe-IV(O-syn)(TMC)(2+) Complex INORGANIC CHEMISTRY Prakash, J., Rohde, G. T., Meier, K. K., Muenck, E., Que, L. 2015; 54 (23): 11055-11057


    Fe(II)(TMC)(OTf)2 reacts with 2-(t)BuSO2-C6H4IO to afford an oxoiron(IV) product, 2, distinct from the previously reported [Fe(IV)(Oanti)(TMC)(NCMe)](2+). In MeCN, 2 has a blue-shifted near-IR band, a higher ν(Fe═O), a larger Mössbauer quadrupole splitting, and quite a distinct (1)H NMR spectrum. Structural analysis of crystals grown from CH2Cl2 reveals a complex with the formulation of [Fe(IV)(Osyn)(TMC)(OTf)](OTf) and the shortest Fe(IV)═O bond [1.625(4) Å] found to date.

    View details for DOI 10.1021/acs.inorgchem.5b02011

    View details for Web of Science ID 000366152500006

    View details for PubMedID 26615667

  • A Long-Lived Fe-III-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mossbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods INORGANIC CHEMISTRY Meier, K. K., Rogers, M. S., Kovaleva, E. G., Mbughuni, M. M., Bominaar, E. L., Lipscomb, J. D., Muenck, E. 2015; 54 (21): 10269-10280
  • Spectroscopic identification of an Fe(III) center, not Fe(IV), in the crystalline Sc-O-Fe adduct derived from [Fe(IV)(O)(TMC)]²?. Journal of the American Chemical Society Prakash, J., Rohde, G. T., Meier, K. K., Jasniewski, A. J., Van Heuvelen, K. M., Münck, E., Que, L. 2015; 137 (10): 3478-3481


    The apparent Sc(3+) adduct of [Fe(IV)(O)(TMC)](2+) (1, TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) has been synthesized in amounts sufficient to allow its characterization by various spectroscopic techniques. Contrary to the earlier assignment of a +4 oxidation state for the iron center of 1, we establish that 1 has a high-spin iron(III) center based on its Mössbauer and EPR spectra and its quantitative reduction by 1 equiv of ferrocene to [Fe(II)(TMC)](2+). Thus, 1 is best described as a Sc(III)-O-Fe(III) complex, in agreement with previous DFT calculations (Swart, M. Chem. Commun. 2013, 49, 6650.). These results shed light on the interaction of Lewis acids with high-valent metal-oxo species.

    View details for DOI 10.1021/jacs.5b00535

    View details for PubMedID 25743366

  • Modeling TauD-J: A High-Spin Nonheme Oxoiron(IV) Complex with High Reactivity toward C-H Bonds JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Biswas, A. N., Puri, M., Meier, K. K., Oloo, W. N., Rohde, G. T., Bominaar, E. L., Muenck, E., Que, L. 2015; 137 (7): 2428-2431


    High-spin oxoiron(IV) species are often implicated in the mechanisms of nonheme iron oxygenases, their C-H bond cleaving properties being attributed to the quintet spin state. However, the few available synthetic S = 2 Fe(IV)═O complexes supported by polydentate ligands do not cleave strong C-H bonds. Herein we report the characterization of a highly reactive S = 2 complex, [Fe(IV)(O)(TQA)(NCMe)](2+) (2) (TQA = tris(2-quinolylmethyl)amine), which oxidizes both C-H and C═C bonds at -40 °C. The oxidation of cyclohexane by 2 occurs at a rate comparable to that of the oxidation of taurine by the TauD-J enzyme intermediate after adjustment for the different temperatures of measurement. Moreover, compared with other S = 2 complexes characterized to date, the spectroscopic properties of 2 most closely resemble those of TauD-J. Together these features make 2 the best electronic and functional model for TauD-J to date.

    View details for DOI 10.1021/ja511757j

    View details for Web of Science ID 000350192700005

    View details for PubMedID 25674662

  • An Unusual Peroxo Intermediate of the Arylamine Oxygenase of the Chloramphenicol Biosynthetic Pathway JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Makris, T. M., Vu, V. V., Meier, K. K., Komor, A. J., Rivard, B. S., Muenck, E., Que, L., Lipscomb, J. D. 2015; 137 (4): 1608-1617


    Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η(1):η(1)) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O-O) that is at least 60 cm(-1) lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η(1):η(1)-peroxo ligand; we propose that a μ-η(1):η(2)-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

    View details for DOI 10.1021/ja511649n

    View details for Web of Science ID 000349138600037

    View details for PubMedID 25564306

  • Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin. Journal of the American Chemical Society Liu, J., Meier, K. K., Tian, S., Zhang, J., Guo, H., Schulz, C. E., Robinson, H., Nilges, M. J., Münck, E., Lu, Y. 2014; 136 (35): 12337-12344


    Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

    View details for DOI 10.1021/ja505410u

    View details for PubMedID 25082811

  • Characterization of a paramagnetic mononuclear nonheme iron-superoxo complex. Journal of the American Chemical Society Chiang, C., Kleespies, S. T., Stout, H. D., Meier, K. K., Li, P., Bominaar, E. L., Que, L., Münck, E., Lee, W. 2014; 136 (31): 10846-10849


    O2 bubbling into a THF solution of Fe(II)(BDPP) (1) at -80 °C generates a reversible bright yellow adduct 2. Characterization by resonance Raman and Mössbauer spectroscopy provides complementary insights into the nature of 2. The former shows a resonance-enhanced vibration at 1125 cm(-1), which can be assigned to the ν(O-O) of a bound superoxide, while the latter reveals the presence of a high-spin iron(III) center that is exchange-coupled to the superoxo ligand, like the Fe(III)-O2(-) pair found for the O2 adduct of 4-nitrocatechol-bound homoprotocatechuate 2,3-dioxygenase. Lastly, 2 oxidizes dihydroanthracene to anthracene, supporting the notion that Fe(III)-O2(-) species can carry out H atom abstraction from a C-H bond to initiate the 4-electron oxidation of substrates proposed for some nonheme iron enzymes.

    View details for DOI 10.1021/ja504410s

    View details for PubMedID 25036460

  • An ultra-stable oxoiron(IV) complex and its blue conjugate base CHEMICAL SCIENCE England, J., Bigelow, J. O., Van Heuvelen, K. M., Farquhar, E. R., Martinho, M., Meier, K. K., Frisch, J. R., Muenck, E., Que, L. 2014; 5 (3): 1204-1215

    View details for DOI 10.1039/c3sc52755g

    View details for Web of Science ID 000332189900042

  • Identification of a low-spin acylperoxoiron(III) intermediate in bio-inspired non-heme iron-catalysed oxidations NATURE COMMUNICATIONS Oloo, W. N., Meier, K. K., Wang, Y., Shaik, S., Muenck, E., Que, L. 2014; 5

    View details for DOI 10.1038/ncomms4046

    View details for Web of Science ID 000331083800029

  • Sc3+-Triggered Oxoiron(IV) Formation from O-2 and its Non-Heme Iron(II) Precursor via a Sc3+-Peroxo-Fe3+ Intermediate JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Li, F., Van Heuvelen, K. M., Meier, K. K., Muenck, E., Que, L. 2013; 135 (28): 10198-10201


    We report that redox-inactive Sc(3+) can trigger O2 activation by the Fe(II)(TMC) center (TMC = tetramethylcyclam) to generate the corresponding oxoiron(IV) complex in the presence of BPh4(-) as an electron donor. To model a possible intermediate in the above reaction, we generated an unprecedented Sc(3+) adduct of [Fe(III)(η(2)-O2)(TMC)](+) by an alternative route, which was found to have an Fe(3+)-(μ-η(2):η(2)-peroxo)-Sc(3+) core and to convert to the oxoiron(IV) complex. These results have important implications for the role a Lewis acid can play in facilitating O-O bond cleavage during the course of O2 activation at non-heme iron centers.

    View details for DOI 10.1021/ja402645y

    View details for Web of Science ID 000322103000005

    View details for PubMedID 23802702

  • Intermediate P* from Soluble Methane Monooxygenase Contains a Diferrous Cluster BIOCHEMISTRY Banerjee, R., Meier, K. K., Muenck, E., Lipscomb, J. D. 2013; 52 (25): 4331-4342


    During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the Fe(II)Fe(II) state (H(red)). O₂ binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(IV) intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of H(red) and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of H(red) and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the Fe(II)Fe(II) state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the Fe(II)Fe(II) center rather than oxidation. The similarity of the optical and Mössbauer spectra of H(red), O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O₂ to be activated for hydrocarbon oxidation.

    View details for DOI 10.1021/bi400182y

    View details for Web of Science ID 000321093900006

    View details for PubMedID 23718184

  • Substrate-Mediated Oxygen Activation by Homoprotocatechuate 2,3-Dioxygenase: Intermediates Formed by a Tyrosine 257 Variant BIOCHEMISTRY Mbughuni, M. M., Meier, K. K., Muenck, E., Lipscomb, J. D. 2012; 51 (44): 8743-8754


    Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O(2) bind in adjacent ligand sites of the active site Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe(II) to O(2) gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases k(cat) by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F(Int1)(HPCA) and Y257F(Int2)(HPCA), accumulate during the Y257F-HPCA + O(2) reaction prior to formation of the ring-cleaved product. Y257F(Int1)(HPCA) is colorless and is formed as O(2) binds reversibly to the HPCA−enzyme complex. Y257F(Int2)(HPCA) forms spontaneously from Y257F(Int1)(HPCA) and displays a chromophore at 425 nm (ε(425) = 10 500 M(−1) cm(−1)). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe(II). The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe(II), and the low O(2) affinity suggest that Y257F(Int1)(HPCA) is an HPCA-Fe(II)-O(2) complex with little electron delocalization onto the O(2). In contrast, the intense spectrum of Y257F(Int2)(HPCA) suggests the intermediate is most likely an HPCA quinone-Fe(II)-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O(2).

    View details for DOI 10.1021/bi301114x

    View details for Web of Science ID 000310664200005

    View details for PubMedID 23066705

  • Protonation of a Peroxodiiron(III) Complex and Conversion to a Diiron(III/IV) Intermediate: Implications for Proton-Assisted O-O Bond Cleavage in Nonheme Diiron Enzymes INORGANIC CHEMISTRY Cranswick, M. A., Meier, K. K., Shan, X., Stubna, A., Kaizer, J., Mehn, M. P., Muenck, E., Que, L. 2012; 51 (19): 10417-10426


    Oxygenation of a diiron(II) complex, [Fe(II)(2)(μ-OH)(2)(BnBQA)(2)(NCMe)(2)](2+) [2, where BnBQA is N-benzyl-N,N-bis(2-quinolinylmethyl)amine], results in the formation of a metastable peroxodiferric intermediate, 3. The treatment of 3 with strong acid affords its conjugate acid, 4, in which the (μ-oxo)(μ-1,2-peroxo)diiron(III) core of 3 is protonated at the oxo bridge. The core structures of 3 and 4 are characterized in detail by UV-vis, Mössbauer, resonance Raman, and X-ray absorption spectroscopies. Complex 4 is shorter-lived than 3 and decays to generate in ~20% yield of a diiron(III/IV) species 5, which can be identified by electron paramagnetic resonance and Mössbauer spectroscopies. This reaction sequence demonstrates for the first time that protonation of the oxo bridge of a (μ-oxo)(μ-1,2-peroxo)diiron(III) complex leads to cleavage of the peroxo O-O bond and formation of a high-valent diiron complex, thereby mimicking the steps involved in the formation of intermediate X in the activation cycle of ribonucleotide reductase.

    View details for DOI 10.1021/ic301642w

    View details for Web of Science ID 000309298200048

    View details for PubMedID 22971084

  • One-electron oxidation of an oxoiron(IV) complex to form an [O=Fe-V=NR](+) center PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Van Heuvelen, K. M., Fiedler, A. T., Shan, X., De Hont, R. F., Meier, K. K., Bominaar, E. L., Muenck, E., Que, L. 2012; 109 (30): 11933-11938


    Oxoiron(V) species are postulated to be involved in the mechanisms of the arene cis-dihydroxylating Rieske dioxygenases and of bioinspired nonheme iron catalysts for alkane hydroxylation, olefin cis-dihydroxylation, and water oxidation. In an effort to obtain a synthetic oxoiron(V) complex, we report herein the one-electron oxidation of the S = 1 complex [Fe(IV)(O)(TMC)(NCCH(3))](2+) (1, where TMC is tetramethylcyclam) by treatment with tert -butyl hydroperoxide and strong base in acetonitrile to generate a metastable complex 2 at -44 °C, which has been characterized by UV-visible, resonance Raman, Mössbauer, and EPR methods. The defining spectroscopic characteristic of 2 is the unusual x/y anisotropy observed for the (57)Fe and (17)O A tensors associated with the high-valent Fe═O unit and for the (14)N A tensor of a ligand derived from acetonitrile. As shown by detailed density functional theory (DFT) calculations, the unusual x/y anisotropy observed can only arise from an iron center with substantially different spin populations in the d(xz) and d(yz) orbitals, which cannot correspond to an Fe(IV)═O unit but is fully consistent with an Fe(V) center, like that found for [Fe(V)(O)(TAML)](-) (where TAML is tetraamido macrocyclic ligand), the only well-characterized oxoiron(V) complex reported. Mass spectral analysis shows that the generation of 2 entails the addition of an oxygen atom to 1 and the loss of one positive charge. Taken together, the spectroscopic data and DFT calculations support the formulation of 2 as an iron(V) complex having axial oxo and acetylimido ligands, namely [Fe(V)(O)(TMC)(NC(O)CH(3))](+).

    View details for DOI 10.1073/pnas.1206457109

    View details for Web of Science ID 000306992700020

    View details for PubMedID 22786933

  • Oxy Intermediates of Homoprotocatechuate 2,3-Dioxygenase: Facile Electron Transfer between Substrates BIOCHEMISTRY Mbughuni, M. M., Chakrabarti, M., Hayden, J. A., Meier, K. K., Dalluge, J. J., Hendrich, M. P., Muenck, E., Lipscomb, J. D. 2011; 50 (47): 10262-10274


    Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD−HPCA and H200N−HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N−HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD−HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mössbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.

    View details for DOI 10.1021/bi201436n

    View details for Web of Science ID 000297143700010

    View details for PubMedID 22011290

  • Characterization of a High-Spin Non-Heme Fe-III-O Intermediate and Its Quantitative Conversion to an Fe-IV = O Complex JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Li, F., Meier, K. K., Cranswick, M. A., Chakrabarti, M., Van Heuvelen, K. M., Muenck, E., Que, L. 2011; 133 (19): 7256-7259


    We have generated a high-spin Fe(III)-OOH complex supported by tetramethylcyclam via protonation of its conjugate base and characterized it in detail using various spectroscopic methods. This Fe(III)-OOH species can be converted quantitatively to an Fe(IV)═O complex via O-O bond cleavage; this is the first example of such a conversion. This conversion is promoted by two factors: the strong Fe(III)-OOH bond, which inhibits Fe-O bond lysis, and the addition of protons, which facilitates O-O bond cleavage. This example provides a synthetic precedent for how O-O bond cleavage of high-spin Fe(III)-peroxo intermediates of non-heme iron enzymes may be promoted.

    View details for DOI 10.1021/ja111742z

    View details for Web of Science ID 000290782200006

    View details for PubMedID 21517091

  • NMR Studies on Domain Diffusion and Alignment in Modular GB1 Repeats BIOPHYSICAL JOURNAL Walsh, J. D., Meier, K., Ishima, R., Gronenborn, A. M. 2010; 99 (8): 2636-2646


    Modular proteins contain individual domains that are often connected by flexible, unstructured linkers. Using a model system based on the GB1 domain, we constructed tandem repeat proteins and investigated the rotational diffusion and long-range angular ordering behavior of individual domains by measuring NMR relaxation parameters and residual dipolar couplings. Although they display almost identical protein-solvent interfaces, each domain exhibits distinct rotational diffusion and alignment properties. The diffusion tensor anisotropy of the N-terminal domain (NTD) is D(‖)/D(⊥) = 1.5-1.6, similar to that of single-GB1 domains (D(‖)/D(⊥) = 1.6-1.7), whereas the value for the C-terminal domain (CTD) is D(‖)/D(⊥) = 2.0-2.2. In addition, the two domains have different rotational correlation times. These effects are observed for linkers of three to 24 residues, irrespective of linker length. The NTD and CTD also differ in their degree of magnetic alignment, even with a flexible linker of 18 residues, exhibiting D(a) values of 7.7 Hz and 9.7 Hz, respectively. Our results suggest that diffusion differences and long-range influences may persist in modular protein systems, even for systems that have highly flexible linkers and exhibit no domain-domain or domain-linker interactions.

    View details for DOI 10.1016/j.bpj.2010.08.036

    View details for Web of Science ID 000283412500031

    View details for PubMedID 20959105