Nuclear Resonance Vibrational Spectroscopy Research Articles

Terminal Hydride Species in [FeFe]‐Hydrogenases Are Vibrationally Coupled to the Active Site Environment

AbstractA combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe−H/D bending modes in CrHydA1 [FeFe]‐hydrogenase Cys‐to‐Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H‐cluster from a ‐SH to ‐OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H‐cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe−H/D normal modes. We observed a significant shift to higher frequency in an Fe−H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)‐substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the ‐OH group of C169S with the bridgehead ‐NH‐ moiety of the active site, as compared to that of the ‐SH group of C169 in the native enzyme.

Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment.

A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We observed a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.

How Does a Heme Carbene Differ from Diatomic Ligated (NO, CO, and CN-) Analogues in the Axial Bond?

Compared to well studied diatomic ligands (NO, CN-, CO), the axial bonds of carbene hemes is much less known although its significance in biological chemistry. The unusually large quadrupole splitting (Δ EQ = +2.2 mm·s-1) and asymmetric parameter (η = 0.9) of the five-coordinate heme carbene [Fe(TTP)(CCl2)], which is the largest among all known low spin ferrohemes, has driven investigations by means of Mössbauer effect Nuclear Resonance Vibrational Spectroscopy (NRVS). Three distinct measurements on one single crystal (two in-plane and one out-of-plane) have demonstrated comprehensive vibrational structures including stretch (429) and bending modes (472 cm-1) of the axial Fe-CCl2, and revealed iron vibrational anisotropy in three orthogonal directions for the first time. Frontier orbital analysis especially comparisons with diatomic analogues (NO, CN-, CO) suggest that CCl2, similar to NO, has led to strong but anisotropic π bonding in a ligand-based "4C"-coordinate which induced the vibrational anisotropies and very large Mössbauer parameters. This is contrasted to CN- and CO complexes which possess a porphyrin-based "4N"-coordinate electronic and vibrational structures due to inherent on-axis linear ligation.

High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT.

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.

High‐Frequency Fe–H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT

AbstractHigh‐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 57Fe nuclear resonance vibrational spectroscopy (NRVS) study on an Fe(μ‐H)2Fe 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.

Atomic force microscopy based infrared (AFM–IR) spectroscopy and nuclear resonance vibrational spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time

Atomic force microscopy based infrared (AFM–IR) spectroscopy and nuclear resonance vibrational spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native FeII O2 Reaction.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII-(hydro)peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII-superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo species in nonheme Fe biochemistry.

Measurement of Substrate Analogue-bound Form of Membrane-integrated Nitric Oxide Reductase by Nuclear Resonance Vibrational Spectroscopy

Measurement of Substrate Analogue-bound Form of Membrane-integrated Nitric Oxide Reductase by Nuclear Resonance Vibrational Spectroscopy

Establishment of Experimental Condition of Nuclear Resonance Vibrational Spectroscopy for Elucidation of Molecular Mechanism of Nitric Oxide Reductase-catalyzed Reaction

Establishment of Experimental Condition of Nuclear Resonance Vibrational Spectroscopy for Elucidation of Molecular Mechanism of Nitric Oxide Reductase-catalyzed Reaction

Application of Nuclear Resonance Vibrational Spectroscopy to Membrane-integrated Nitric Oxide Reductase

Application of Nuclear Resonance Vibrational Spectroscopy to Membrane-integrated Nitric Oxide Reductase

NRVS for Fe in Biology: Experiment and Basic Interpretation.

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.

Iron(III, IV, V)-oxo complexes studied by nuclear resonance vibrational spectroscopy

Iron(III, IV, V)-oxo complexes studied by nuclear resonance vibrational spectroscopy

Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior to H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

What Can Be Learned from Nuclear Resonance Vibrational Spectroscopy: Vibrational Dynamics and Hemes.

Nuclear resonance vibrational spectroscopy (NRVS; also known as nuclear inelastic scattering, NIS) is a synchrotron-based method that reveals the full spectrum of vibrational dynamics for Mössbauer nuclei. Another major advantage, in addition to its completeness (no arbitrary optical selection rules), is the unique selectivity of NRVS. The basics of this recently developed technique are first introduced with descriptions of the experimental requirements and data analysis including the details of mode assignments. We discuss the use of NRVS to probe 57Fe at the center of heme and heme protein derivatives yielding the vibrational density of states for the iron. The application to derivatives with diatomic ligands (O2, NO, CO, CN–) shows the strong capabilities of identifying mode character. The availability of the complete vibrational spectrum of iron allows the identification of modes not available by other techniques. This permits the correlation of frequency with other physical properties. A significant example is the correlation we find between the Fe–Im stretch in six-coordinate Fe(XO) hemes and the trans Fe–N(Im) bond distance, not possible previously. NRVS also provides uniquely quantitative insight into the dynamics of the iron. For example, it provides a model-independent means of characterizing the strength of iron coordination. Prediction of the temperature-dependent mean-squared displacement from NRVS measurements yields a vibrational “baseline” for Fe dynamics that can be compared with results from techniques that probe longer time scales to yield quantitative insights into additional dynamical processes.

Ferric Heme-Nitrosyl Complexes: Kinetically Robust or Unstable Intermediates?

We have determined a convenient method for the bulk synthesis of high-purity ferric heme-nitrosyl complexes ({FeNO}6 in the Enemark-Feltham notation); this method is based on the chemical or electrochemical oxidation of corresponding {FeNO}7 precursors. We used this method to obtain the five- and six-coordinate complexes [Fe(TPP)(NO)]+ (TPP2- = tetraphenylporphyrin dianion) and [Fe(TPP)(NO)(MI)]+ (MI = 1-methylimidazole) and demonstrate that these complexes are stable in solution in the absence of excess NO gas. This is in stark contrast to the often-cited instability of such {FeNO}6 model complexes in the literature, which is likely due to the common presence of halide impurities (although other impurities could certainly also play a role). This is avoided in our approach for the synthesis of {FeNO}6 complexes via oxidation of pure {FeNO}7 precursors. On the basis of these results, {FeNO}6 complexes in proteins do not show an increased stability toward NO loss compared to model complexes. We also prepared the halide-coordinated complexes [Fe(TPP)(NO)(X)] (X = Cl-, Br-), which correspond to the elusive, key reactive intermediate in the so-called autoreduction reaction, which is frequently used to prepare {FeNO}7 complexes from ferric precursors. All of the complexes were characterized using X-ray crystallography, UV-vis, IR, and nuclear resonance vibrational spectroscopy (NRVS). On the basis of the vibrational data, further insight into the electronic structure of these {FeNO}6 complexes, in particular with respect to the role of the axial ligand trans to NO, is obtained.

A Combined Probe-Molecule, Mössbauer, Nuclear Resonance Vibrational Spectroscopy, and Density Functional Theory Approach for Evaluation of Potential Iron Active Sites in an Oxygen Reduction Reaction Catalyst

Nonprecious metal M–N–C (M = Fe or Co) catalysts that are effective for the oxygen-reduction reaction in polymer-electrolyte fuel cells have been developed, but no consensus has yet been reached regarding the nature of the M sites in these heterogeneous catalysts that are responsible for the reaction with dioxygen (O2). While multiple studies have developed correlations between Fe distributions in as-prepared catalysts and ORR activity, the direct identification of sites reactive toward O2 or O2-analogue molecules remains a significant challenge. In the present study, we demonstrate a new approach to identifying and characterizing potential Fe active sites in complex ORR catalysts that combines an effective probe molecule (NO(g)), Mossbauer spectroscopy, and nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations. Mossbauer spectroscopic studies demonstrate that NO(g) treatment of electrochemically reduced PANI–57Fe–C leads to a selective reaction with only a su...

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy.

[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.

Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an example

1H spin-lattice Nuclear Magnetic Resonance (NMR) relaxometry, vibrational spectroscopy and Atomic Force Microscopy (AFM) have been applied to differentiate between original and counterfeit Viagra®. The relaxation studies have been performed in a frequency range covering four orders of magnitude, from 4kHz to 40MHz. It has been shown that for the counterfeit product the relaxation is bi-exponential in the whole frequency range, while for the original Viagra® the relaxation process is always single exponential. Thus, even a qualitative analysis of the relaxation data makes it possible to identify the falsified medicine. Moreover, it has been demonstrated that vibrational spectroscopy does not allow for differentiating between the products, while AFM studies are likely to lead one to deceptive conclusions regarding the originality of the medicine. Furthermore, a quantitative analysis of the relaxation data has been performed to describe in detail the relaxation properties of the original and falsified products.

High-resolution monochromator for iron nuclear resonance vibrational spectroscopy of biological samples

A new high-resolution monochromator for 14.4-keV X-rays has been designed and developed for the Fe nuclear resonance vibrational spectroscopy of biological samples. In addition to high resolution, higher flux and stability are especially important for measuring biological samples, because of the very weak signals produced due to the low concentrations of Fe-57. A 24% increase in flux while maintaining a high resolution better than 0.9 meV is achieved in the calculation by adopting an asymmetric reflection of Ge, which is used as the first crystal of the three-bounce high-resolution monochromator. A 20% increase of the exit beam size is acceptable to our biological applications. The higher throughput of the new design has been experimentally verified. A fine rotation mechanics that combines a weak-link hinge with a piezoelectric actuator was used for controlling the photon energy of the monochromatic beam. The resulting stability is sufficient to preserve the intrinsic resolution.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.

The reaction of protein‐bound iron–sulfur (Fe‐S) clusters with nitric oxide (NO) plays key roles in NO‐mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe‐S clusters has been hampered by a lack of information about the nature of the iron‐nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe‐4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2(NO)4(Cys)2]) and Roussin's Black Salt (RBS, [Fe4(NO)7S3]. In the latter case, the absence of 32S/34S shifts in the Fe−S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.