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ABSTRACT: Azotobacter vinelandii nitrogenase Fe protein (Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies with DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10-30 cm-1 decrease in Fe-S stretching frequencies with each added electron. The "oxidized" [4Fe-4S]2+ state spectrum is consistent with and extends previous resonance Raman spectra. For the "reduced" [4Fe-4S]1+ state in Fe protein, and for any "all-ferrous" [4Fe-4S]0 cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe-S and Fe-Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized Av2, EXAFS and DFT indicate nearly equal Fe-Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to k = 21 Å-1 indicates a 1:1 ratio of Fe-Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe-S and Fe-Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the Av2 protein environment.
Journal of the American Chemical Society 01/2013; · 9.91 Impact Factor
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Jon M Kuchenreuther, Yisong Guo,
Hongxin Wang,
William K Myers,
Simon J George,
Christine A Boyke,
Yoshitaka Yoda,
Esen Ercan Alp,
Jiyong Zhao,
R David Britt,
James R Swartz,
Stephen P Cramer
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ABSTRACT: The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four [FeS] clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. In order to enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled [FeS] clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second [(57)FeS] cluster. EPR spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.
Biochemistry 12/2012; · 3.42 Impact Factor
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ABSTRACT: Nucleophilic oxidant: The reaction between a thiolato iron(II) complex 1 and superoxide in aprotic solvent at -90 °C yields a novel thiolato iron(III) peroxide intermediate 2, which exhibits unusually high nucleophilic reactivity. Compound 2 is an isomer of the thiolato iron(II) superoxide intermediate that is invoked in the reaction between superoxide reductase and superoxide.
Angewandte Chemie International Edition 08/2012; 51(36):9132-6. · 13.45 Impact Factor
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ABSTRACT: IscR is an Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mössbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S](+) cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the [2Fe-2S](2+) form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and nuclear magnetic resonance spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved His107 residue is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.
Biochemistry 05/2012; 51(22):4453-62. · 3.42 Impact Factor
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ABSTRACT: We have used EXAFS and NRVS spectroscopies to examine the structural changes in the FeMo-cofactor active site of the α-70(Ala) variant of Azotobacter vinelandii nitrogenase on binding and reduction of propargyl alcohol (PA). The Mo K-edge near-edge and EXAFS spectra are very similar in the presence and absence of PA, suggesting PA does not bind at Mo. By contrast, Fe EXAFS spectra show a clear and reproducible change in the long Fe-Fe interaction at ~3.7 Å on PA binding with the apparent appearance of a new Fe-Fe interaction at 3.99 Å. An analogous change in the long Mo-Fe 5.1 Å interaction is not seen. The NRVS spectra exclude the possibility of large-scale structural change of the FeMo-cofactor involving breaking the μ(2) Fe-S-Fe bonds of the Fe(6)S(9)X core. The simplest chemically consistent structural change is that the bound form of PA is coordinated at Fe atoms (Fe6 or Fe7) adjacent to the Mo terminus, with a concomitant movement of the Fe away from the central atom X and along the Fe-X bond by about 0.35 Å. This study comprises the first experimental evidence of the conformational changes of the FeMo-cofactor active site on binding a substrate or product.
Journal of inorganic biochemistry 02/2012; 112:85-92. · 3.25 Impact Factor
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ABSTRACT: The pentadentate ligand (n)Bu-P2DA (2(b), (n)Bu-P2DA = N-(1',1'-bis(2-pyridyl)pentyl)iminodiacetate) was designed to bind an iron center in a carboxylate-rich environment similar to that found in the active sites of TauD and other α-ketoglutarate-dependent mononuclear non-heme iron enzymes. The iron(II) complex (n)Bu(4)N[Fe(II)(Cl)((n)Bu-P2DA)] (3(b)-Cl) was synthesized and crystallographically characterized to have a 2-pyridine-2-carboxylate donor set in the plane perpendicular to the Fe-Cl bond. Reaction of 3(b)-Cl with N-heterocyclic amines such as pyridine or imidazole yielded the N-heterocyclic amine adducts [Fe(II)(N)((n)Bu-P2DA)]. These adducts in turn reacted with oxo-transfer reagents at -95 °C to afford a short-lived oxoiron(IV) complex [Fe(IV)(O)((n)Bu-P2DA)] (5(b)) in yields as high as 90% depending on the heterocycle used. Complex 5(b) exhibits near-IR absorption features (λ(max) = 770 nm) and Mossbauer parameters (δ = 0.04 mm/s; ΔE(Q) = 1.13 mm/s; D = 27±2 cm(-1)) characteristic of an S = 1 oxoiron(IV) species. Direct evidence for an Fe=O bond of 1.66 Å was found from EXAFS analysis. DFT calculations on 5(b) in its S =1 spin state afforded a geometry-optimized structure consistent with the EXAFS data. They further demonstrated that the replacement of two pyridine donors in [Fe(IV)(O)(N4Py)](2+) (N4Py = N,N-(bis(2-pyridyl)methyl)N-bis(2-pyridylmethyl)amine) with carboxylate donors in 5(b) decreased the energy gap between the ground S = 1 and the excited S = 2 states, reflecting the weaker equatorial ligand field of 5(b) and accounting for its larger D value. Complex 5(b) reacted readily with dihydrotoluene, methyldiphenylphosphine and ferrocene at -60 °C, and in all cases was approximately a 5-fold more reactive oxidant than [Fe(IV)(O)(N4Py)](2+). The reactivity differences between these two complexes may arise from a combination of electronic and steric factors. Carboxylate-rich 5(b) represents the closest structural mimic reported thus far of the oxoiron(IV) intermediate ('J') found in TauD and provides us with vital insights into the role carboxylate ligands play in modulating the spectroscopic and reactivity properties of the non-heme oxoiron(IV) moiety.
Chemical science (Royal Society of Chemistry) 01/2012; 3:1680-1693. · 7.53 Impact Factor
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ABSTRACT: The trigonal-bipyramidal high-spin (S = 2) oxoiron(IV) complex [Fe(IV)(O)(TMG(2)dien)(CH(3)CN)](2+) (7) was synthesized and spectroscopically characterized. Substitution of the CH(3)CN ligand by anions, demonstrated here for X = N(3)(-) and Cl(-), yielded additional S = 2 oxoiron(IV) complexes of general formulation [Fe(IV)(O)(TMG(2)dien)(X)](+) (7-X). The reduced steric bulk of 7 relative to the published S = 2 complex [Fe(IV)(O)(TMG(3)tren)](2+) (2) was reflected by enhanced rates of intermolecular substrate oxidation.
Journal of the American Chemical Society 08/2011; 133(31):11880-3. · 9.91 Impact Factor
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Devrani Mitra,
Vladimir Pelmenschikov, Yisong Guo,
David A Case,
Hongxin Wang,
Weibing Dong,
Ming-Liang Tan,
Toshiko Ichiye,
Francis E Jenney,
Michael W W Adams,
Yoshitaka Yoda,
Jiyong Zhao,
Stephen P Cramer
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ABSTRACT: We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
Biochemistry 06/2011; 50(23):5220-35. · 3.42 Impact Factor
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ABSTRACT: Currently, there are only a handful of synthetic S = 2 oxoiron(IV) complexes. These serve as models for the high-spin (S = 2) oxoiron(IV) species that have been postulated, and confirmed in several cases, as key intermediates in the catalytic cycles of a variety of nonheme oxygen activating enzymes. The trigonal bipyramidal complex [Fe(IV)(O)(TMG(3)tren)](2+) (1) was both the first S = 2 oxoiron(IV) model complex to be generated in high yield and the first to be crystallographically characterized. In this study, we demonstrate that the TMG(3)tren ligand is also capable of supporting a tricationic cyanoiron(IV) unit, [Fe(IV)(CN)(TMG(3)tren)](3+) (4). This complex was generated by electrolytic oxidation of the high-spin (S = 2) iron(II) complex [Fe(II)(CN)(TMG(3)tren)](+) (2), via the S = 5/2 complex [Fe(III)(CN)(TMG(3)tren)](2+) (3), the progress of which was conveniently monitored by using UV-vis spectroscopy to follow the growth of bathochromically shifting ligand-to-metal charge transfer (LMCT) bands. A combination of X-ray absorption spectroscopy (XAS), Mössbauer and NMR spectroscopies was used to establish that 4 has a S = 0 iron(IV) center. Consistent with its diamagnetic iron(IV) ground state, extended X-ray absorption fine structure (EXAFS) analysis of 4 indicated a significant contraction of the iron-donor atom bond lengths, relative to those of the crystallographically characterized complexes 2 and 3. Notably, 4 has an Fe(IV/III) reduction potential of ∼1.4 V vs Fc(+/o), the highest value yet observed for a monoiron complex. The relatively high stability of 4 (t(1/2) in CD(3)CN solution containing 0.1 M KPF(6) at 25 °C ≈ 15 min), as reflected by its high-yield accumulation via slow bulk electrolysis and amenability to (13)C NMR at -40 °C, highlights the ability of the sterically protecting, highly basic peralkylguanidyl donors of the TMG(3)tren ligand to support highly charged high-valent complexes.
Inorganic Chemistry 03/2011; 50(7):2885-96. · 4.60 Impact Factor
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ABSTRACT: Infrared, Raman, and nuclear resonant vibrational (NRVS) spectroscopies have been used to address the Fe-H bonding in trans-HFe(CO) iron hydride compound, HFe(CO)(dppe)2, dppe = 1,2-bis(diphenylphosphino)ethane. H and D isotopomers of the compound, with selective substitution at the metal-coordinated hydrogen, have been considered in order to address the Fe-H/D stretching and bending modes. Experimental results are compared to the normal mode analysis by density functional theory (DFT). The results are that (i) the IR spectrum does not clearly show Fe-H stretching or bending modes; (ii) Fe-H stretching modes are clear but weak in the Raman spectrum, and Fe-H bending modes are weak; (iii) NRVS 57Fe spectroscopy resolves Fe-H bending clearly, but Fe-H or Fe-D stretching is above its experimentally resolved frequency range. DFT calculations (with no scaling of frequencies) show intensities and peak locations that allow unambiguous correlations between observed and calculated features, with frequency errors generally less than 15 cm(-1). Prospects for using these techniques to unravel vibrational modes of protein active sites are discussed.
Faraday Discussions 01/2011; 148:409-20; discussion 421-41. · 5.00 Impact Factor
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ABSTRACT: The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mossbauer and electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished approximately 3-fold, while non-heme HS Fe(II) species, non-heme mononuclear HS Fe(III), and Fe(III) nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS Fe(II) ions serves as feedstock for Fe-S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.
Biochemistry 07/2010; 49(26):5436-44. · 3.42 Impact Factor
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ABSTRACT: [Fe(IV)(O)(TMG(3)tren)](2+) (1; TMG(3)tren = 1,1,1-tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine) is a unique example of an isolable synthetic S = 2 oxoiron(IV) complex, which serves as a model for the high-valent oxoiron(IV) intermediates observed in nonheme iron enzymes. Congruent with DFT calculations predicting a more reactive S = 2 oxoiron(IV) center, 1 has a lifetime significantly shorter than those of related S = 1 oxoiron(IV) complexes. The self-decay of 1 exhibits strictly first-order kinetic behavior and is unaffected by solvent deuteration, suggesting an intramolecular process. This hypothesis was supported by ESI-MS analysis of the iron products and a significant retardation of self-decay upon use of a perdeuteromethyl TMG(3)tren isotopomer, d(36)-1 (KIE = 24 at 25 degrees C). The greatly enhanced thermal stability of d(36)-1 allowed growth of diffraction quality crystals for which a high-resolution crystal structure was obtained. This structure showed an Fe horizontal lineO unit (r = 1.661(2) A) in the intended trigonal bipyramidal geometry enforced by the sterically bulky tetramethylguanidinyl donors of the tetradentate tripodal TMG(3)tren ligand. The close proximity of the methyl substituents to the oxoiron unit yielded three symmetrically oriented short C-D...O nonbonded contacts (2.38-2.49 A), an arrangement that facilitated self-decay by rate-determining intramolecular hydrogen atom abstraction and subsequent formation of a ligand-hydroxylated iron(III) product. EPR and Mossbauer quantification of the various iron products, referenced against those obtained from reaction of 1 with 1,4-cyclohexadiene, allowed formulation of a detailed mechanism for the self-decay process. The solution of this first crystal structure of a high-spin (S = 2) oxoiron(IV) center represents a fundamental step on the path toward a full understanding of these pivotal biological intermediates.
Journal of the American Chemical Society 06/2010; 132(25):8635-44. · 9.91 Impact Factor
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Angewandte Chemie International Edition 05/2010; 49(23):3912-5. · 13.45 Impact Factor
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ABSTRACT: Mössbauer spectroscopy was used to detect pools of Fe in mitochondria from fermenting yeast cells, including those consisting of nonheme high-spin (HS) Fe(II) species, Fe(III) nanoparticles, and mononuclear HS Fe(III) species. At issue was whether these species were located within mitochondria or on their exterior. None could be removed by washing mitochondria extensively with ethylene glycol tetraacetic acid or bathophenanthroline sulfonate (BPS), Fe(II) chelators that do not appear to penetrate mitochondrial membranes. However, when mitochondrial samples were sonicated, BPS coordinated the Fe(II) species, forming a low-spin Fe(II) complex. This treatment also diminished the levels of both Fe(III) species, suggesting that all of these Fe species are encapsulated by mitochondrial membranes and are protected from chelation until membranes are disrupted. 1,10-Phenanthroline is chemically similar to BPS but is membrane soluble; it coordinated nonheme HS Fe(II) in unsonicated mitochondria. Further, the HS Fe(III) species and nanoparticles were not reduced by dithionite until the detergent deoxycholate was added to disrupt membranes. There was no correlation between the percentage of nonheme HS Fe(II) species in mitochondrial samples and the level of contaminating proteins. These results collectively indicate that the observed Fe species are contained within mitochondria. Mossbauer spectra of whole cells were dominated by HS Fe(III) features; the remainder displayed spectral features typical of isolated mitochondria, suggesting that the Fe in fermenting yeast cells can be coarsely divided into two categories: mitochondrial Fe and (mostly) HS Fe(III) ions in one or more non-mitochondrial locations.
Biochemistry 05/2010; 49(19):4227-34. · 3.42 Impact Factor
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ABSTRACT: We have used nuclear resonance vibrational spectroscopy (NRVS) to examine the nature of the Fe-S unit. Specifically, vibrational
characteristics have been determined, and through incremental steps in model system complexity, applied to analysis of the
enzyme nitrogenase. This stepwise strategy demonstrates NRVS as a viable bioinorganic tool, and will undoubtedly increase
the application of synchrotron spectroscopy to biological problems.
Key wordsferredoxin-rubredoxin-vibrational spectroscopy-synchrotron-Mössbauer
04/2010: pages 47-54;
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Yuming Xiao,
Ming-Liang Tan,
Toshiko Ichiye,
Hongxin Wang, Yisong Guo,
Matt C Smith,
Jacques Meyer,
Wolfgang Sturhahn,
Ercan E Alp,
Jiyong Zhao,
Yoshitaka Yoda,
Stephen P Cramer
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ABSTRACT: We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S'(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
Biochemistry 06/2008; 47(25):6612-27. · 3.42 Impact Factor
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Yisong Guo,
Hongxin Wang,
Yuming Xiao,
Sonja Vogt,
Rudolf K Thauer,
Seigo Shima,
Phillip I Volkers,
Thomas B Rauchfuss,
Vladimir Pelmenschikov,
David A Case,
Ercan E Alp,
Wolfgang Sturhahn,
Yoshitaka Yoda,
Stephen P Cramer
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ABSTRACT: We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron site in the iron-sulfur cluster-free hydrogenase Hmd from the methanogenic archaeon Methanothermobacter marburgensis. The spectra have been interpreted by comparison with a cis-(CO)2-ligated Fe model compound, Fe(S2C2H4)(CO)2(PMe3)2, as well as by normal mode simulations of plausible active site structures. For this model complex, normal mode analyses both from an optimized Urey-Bradley force field and from complementary density functional theory (DFT) calculations produced consistent results. For Hmd, previous IR spectroscopic studies found strong CO stretching modes at 1944 and 2011 cm(-1), interpreted as evidence for cis-Fe(CO)2 ligation. The NRVS data provide further insight into the dynamics of the Fe site, revealing Fe-CO stretch and Fe-CO bend modes at 494, 562, 590, and 648 cm(-1), consistent with the proposed cis-Fe(CO)2 ligation. The NRVS also reveals a band assigned to Fe-S stretching motion at approximately 311 cm(-1) and another reproducible feature at approximately 380 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) for Hmd can be reasonably well simulated by a normal mode analysis based on a Urey-Bradley force field for a five-coordinate cis-(CO)2-ligated Fe site with additional cysteine, water, and pyridone cofactor ligands. A "truncated" model without a water ligand can also be used to match the NRVS data. A final interpretation of the Hmd NRVS data, including DFT analysis, awaits a three-dimensional structure for the active site.
Inorganic Chemistry 06/2008; 47(10):3969-77. · 4.60 Impact Factor
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Yuming Xiao,
Ming-Liang Tan,
Toshiko Ichiye,
Hongxin Wang, Yisong Guo,
Matt C. Smith,
Jacques Meyer,
Wolfgang Sturhahn,
Ercan E. Alp,
Jiyong Zhao,
Yoshitaka Yoda,
Stephen P. Cramer
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ABSTRACT: We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe2S2(Cys)4 sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe−S stretching modes peaking around 335 cm−1, and additional features observed as high as the B2u mode at 421 cm−1. Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B3u mode at 288 cm−1, attributed to an asymmetric combination of the two FeS4 breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS4 breathing. NRVS and RR analysis of isotope shifts with 36S-substituted into bridging S2− ions in Rc FdVI allowed quantitation of S2− motion in different normal modes. We observed the symmetric Fe−Fe stretching mode at 190 cm−1 in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm−1. The 57Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey−Bradley force fields. Progressively more complex D2h Fe2S2S′4, C2h Fe2S2(SCC)4, and C1 Fe2S2(Cys)4 models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe−S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe−S proteins.
05/2008;
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Taras Petrenko,
Serena DeBeer George,
Núria Aliaga-Alcalde,
Eckhard Bill,
Bernd Mienert,
Yuming Xiao, YiSong Guo,
Wolfgang Sturhahn,
Stephen P Cramer,
Karl Wieghardt,
Frank Neese
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ABSTRACT: The characterization of high-valent iron species is of interest due to their relevance to biological reaction mechanisms. Recently, we have synthesized and characterized an [Fe(V)-nitrido-cyclam-acetato]+ complex, which has been characterized by Mössbauer, magnetic susceptibility data, and XAS spectroscopies combined with DFT calculations (Aliaga-Alcade, N.; DeBeer George, S.; Bill, E.; Wieghardt, K.; Neese, F. Angew. Chem., Int. Ed. 2005, 44, 2908-2912). The results of this study indicated that the [Fe(V)-nitrido-cyclam-acetato]+ complex is an unusual d3 system with a nearly orbitally degenerate S=1/2 ground state. Although the calculations predicted fairly different Fe-N stretching frequencies for the S=1/2 and the competing S=3/2 ground states, a direct experimental determination of this important fingerprint quantity was missing. Here we apply synchrotron-based nuclear resonance vibrational scattering (NRVS) to characterize the Fe-N stretching frequency of an Fe(V)-nitrido complex and its Fe(III)-azide precursor. The NRVS data show a new isolated band at 864 cm(-1) in the Fe(V)-nitrido complex that is absent in the precursor. The NRVS spectra are fit and simulated using a DFT approach, and the new feature is unambiguously assigned to a Fe(V)-N stretch. The calculated Fe-N stretching frequency is too high by approximately 75 cm(-1). Anharmonic contributions to the Fe-N stretching frequency have been evaluated and have been found to be small (-5.5 cm(-1)). The NRVS data provided a unique opportunity to obtain this vibrational information, which had eluded characterization by more traditional vibrational spectroscopies.
Journal of the American Chemical Society 10/2007; 129(36):11053-60. · 9.91 Impact Factor
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Taras Petrenko,
Serena DeBeer George,
Nuria Aliaga-Alcalde,
Eckhard Bill,
Bernd Mienert,
Yuming Xiao, Yisong Guo,
Wolfgang Sturhahn,
Stephen P Cramer,
Karl Wieghardt,
F. Neese
Journal of the American Chemical Society, v.129, 11053-11060 (2007).
