The geometry of reactive intermediates by nutation NMR spectroscopy: the tert-butyl cation (original) (raw)
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Proceedings of the National Academy of Sciences, 2013
High-valent intermediates of binuclear nonheme iron enzymes are structurally unknown despite their importance for understanding enzyme reactivity. Nuclear resonance vibrational spectroscopy combined with density functional theory calculations has been applied to structurally well-characterized high-valent mono- and di-oxo bridged binuclear Fe model complexes. Low-frequency vibrational modes of these high-valent diiron complexes involving Fe motion have been observed and assigned. These are independent of Fe oxidation state and show a strong dependence on spin state. It is important to note that they are sensitive to the nature of the Fe 2 core bridges and provide the basis for interpreting parallel nuclear resonance vibrational spectroscopy data on the high-valent oxo intermediates in the binuclear nonheme iron enzymes.
Spectroscopic Studies of the Anaerobic Enzyme−Substrate Complex of Catechol 1,2-Dioxygenase
Journal of the American Chemical Society, 2005
The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/ substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm -1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm -1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution. (1) Bugg, T. D. H.; Lin, G. Chem. Commun. 2001, 941-952. (2) Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L. Chem. ReV. 2004, 104, 939-986. (3) Bugg, T. D. H. Tetrahedron 2003, 59, 7075-7101. (4) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S. K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y. S.; Zhou, J.
Biochemical and Biophysical Research Communications, 1980
Resonance enhancement of high and low frequency vibrational modes of oxidized cytochrome ~3 in Several cytochrome oxidase species have been observed when flowing samples and excitation frequencies of 413.1 nm and 406.7 nm were used. The data are interpreted by excitation profile arguments which incorporate optical parameters from the spectra of cytochromes a and a~ deduced by Vanneste (Biochemistry 5, 838-848 (1966)). The carbonyl--of oxT~ized cytochrome a3 is conjugated with the heme a ~-electron system independent of both its iron spln state and the redox state ~f cytochrome a. This contrasts with the behavior of cytochrome a and of reduced cytochr~me ~3" Mechanistic implications of these conformation~l differences are briefly considered.
Accounts of Chemical Research, 2013
O ver the past decades metalloenzymes and their synthetic models have emerged as an area of increasing research interest. The metalloenzymes and their synthetic models oxidize organic molecules using oxometal complexes (OMCs), especially oxoiron(IV)-based ones. Theoretical studies have helped researchers to characterize the active species and to resolve mechanistic issues. This activity has generated massive amounts of data on the relationship between the reactivity of OMCs and the transition metal's identity, oxidation state, ligand sphere, and spin state. Theoretical studies have also produced information on transition state (TS) structures, reaction intermediates, barriers, and rateÀequilibrium relationships. For example, the experi-mentalÀtheoretical interplay has revealed that nonheme enzymes carry out H-abstraction from strong CÀH bonds using high-spin (S = 2) oxoiron(IV) species with four unpaired electrons on the iron center. However, other reagents with higher spin states and more unpaired electrons on the metal are not as reactive. Still other reagents carry out these transformations using lower spin states with fewer unpaired electrons on the metal. The TS structures for these reactions exhibit structural selectivity depending on the reactive spin states. The barriers and thermodynamic driving forces of the reactions also depend on the spin state. H-Abstraction is preferred over the thermodynamically more favorable concerted insertion into CÀH bonds. Currently, there is no unified theoretical framework that explains the totality of these fascinating trends.
Journal of The American Chemical Society, 2006
Crystalline samples of four low-spin Fe(III) octaalkyltetraphenylporphyrinate and two low-spin Fe(III) tetramesitylporphyrinate complexes, all of which are models of the bis-histidine-coordinated cytochromes of mitochondrial complexes II, III, and IV and chloroplast complex b6 f, and whose molecular structures and EPR spectra have been reported previously, have been investigated in detail by Mö ssbauer spectroscopy. The six complexes and the dihedral angles between axial ligand planes of each are [(TMP)- , and perp-[(OMTPP)Fe(1-MeIm)2]Cl (90°). Of these, the first three have been shown to exhibit normal rhombic EPR spectra, each with three clearly resolved g-values, while the last three have been shown to exhibit "large gmax" EPR spectra at 4.2 K. It is found that the hyperfine coupling constants of the complexes are consistent with those reported previously for low-spin ferriheme systems, with the largest-magnitude hyperfine coupling constant, Azz, being considerably smaller for the "parallel" complexes (400-540 kG) than for the strictly perpendicular complex (902 kG), Axx being negative for all six complexes, and Azz and Axx being of similar magnitude for the "parallel" complexes (for example, for [(TMP)Fe(1-MeIm)2]Cl, Azz ) 400 kG, Axx ) -400 kG). In all cases, Ayy is small but difficult to estimate with accuracy. With results for six structurally characterized model systems, we find for the first time qualitative correlations of gzz, Azz, and ∆EQ with axial ligand plane dihedral angle ∆ .
The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b 6 f with the electron transfer proteins plastocyanin and cytochrome c 6 . A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c 6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c 6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c 6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.
Biochemistry, 1999
T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1 H NMR studies of oxidized and reduced T4MOC London] revealed the presence of hyperfine-shifted 1 H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1 H, 2 H, and 15 N signals from T4MOC. Selective deuteration led to identification of signals from the four H atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the H protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1 H spectrum of reduced T4MOC was assigned on the basis of selective 2 H labeling to the His H 1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1 H-13 C correlation (at 25.39 ppm 1 H and 136.11 ppm 13 C) observed in spectra of [U-13 C]T4MOC with a 1 H-13 C coupling constant of ∼192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1 H 1 -13 C 1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1 H R -13 C R . The His H 1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1 H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1 H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Selective 15 N labeling was used to identify hyperfine-shifted 15 N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the N 2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the N δ1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family. centers are found in the cytochrome oxidase complexes (1), in the oxygenase components of enzymes such as the benzene, naphthalene, phthalate, and toluene dioxygenases (2), and in small M r ferredoxins that act as soluble electron carriers for a variety of bacterial oxygenases (3). shows the metal-binding motif C-X-H-X 15-21 -C-X 2 -H that has been identified in all Rieske proteins (2). Spectroscopic studies have revealed an asymmetric iron-sulfur core with the S γ atom of each of the two cysteine residues coordinated to one iron site and with the N δ1 atom of each of the two histidine residues coordinated to the other iron site (4-9). Mössbauer and ENDOR studies have shown that the His-coordinated iron becomes a localized ferrous site upon reduction (4-6).
Biophysical Journal, 1995
The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 104, 342 x 104, and 353 x 104 cm-1, respectively, and are temperature invariant over the temperature ranges studied. An El value at 4 K of 73 x 1 04 cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrin derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the El value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2) zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt care interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-' is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The El value is approximately 60 x 104 cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of -40 cm-1 is estimated. In contrast to those for the metal cytochromes, the El value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.