Sulfur X-ray Absorption and Emission Spectroscopy of Organic Sulfones.

The sulfones are a widespread group of organo-sulfur compounds, which contain the sulfonyl SO2 group attached to two carbons and have a formal sulfur oxidation state of +2. We have examined the sulfur K near-edge X-ray absorption spectroscopy (XAS) of a range of different sulfones and find substantial spectroscopic variability depending upon the nature of the coordination to the sulfonyl group. We have also examined the sulfur Kß X-ray emission spectroscopy (XES) of selected representative sulfones. Density functional theory simulations show satisfactory reproduction of both absorption and emission spectra while enabling assignment of the various transitions comprising the spectra. The correspondence between observed and simulated spectra shows promise for ab initio prediction of sulfur X-ray absorption and emission spectra of sulfones of any substituent. The absorption spectra and, to a lesser extent, the emission spectra are sensitive to the nature of the organic groups bound to the sulfonyl (SO2) moiety, clearly showing the potential of X-ray spectroscopy as an in situ probe of sulfone chemistry.

Nitrogenase Chemistry at 10 Kelvin─Phototautomerization and Recombination of CO-Inhibited α-H195Q Enzyme.

CO-bound forms of nitrogenase are N2-reduction inhibited and likely intermediates in Fischer-Tropsch chemistry. Visible-light photolysis at 7 K was used to interrogate all three known CO-related EPR-active forms as exhibited by the α-H195Q variant of Azotobacter vinelandii nitrogenase MoFe protein. The hi(5)-CO EPR signal converted to the hi-CO EPR signal, which reverted at 10 K. FT-IR monitoring revealed an exquisitely light-sensitive "Hi-2" species with bands at 1932 and 1866 cm-1 that yielded "Hi-1" with bands at 1969 and 1692 cm-1, which reverted to "Hi-2". The similarities of photochemical behavior and recombination kinetics showed, for the first time, that hi-CO EPR and "Hi-1" IR signals arise from one chemical species. hi(5)-CO EPR and "Hi-2" IR signals are from a second species, and lo-CO EPR and "Lo-2" IR signals, formed after prolonged illumination, are from a third species. Comparing FT-IR data with CO-inhibited MoFe-protein crystal structures allowed assignment of CO-bonding geometries in these species.

Abridged spectral matrix inversion: parametric fitting of X-ray fluorescence spectra following integrative data reduction.

Recent improvements in both X-ray detectors and readout speeds have led to a substantial increase in the volume of X-ray fluorescence data being produced at synchrotron facilities. This in turn results in increased challenges associated with processing and fitting such data, both temporally and computationally. Herein an abridging approach is described that both reduces and partially integrates X-ray fluorescence (XRF) data sets to obtain a fivefold total improvement in processing time with negligible decrease in quality of fitting. The approach is demonstrated using linear least-squares matrix inversion on XRF data with strongly overlapping fluorescent peaks. This approach is applicable to any type of linear algebra based fitting algorithm to fit spectra containing overlapping signals wherein the spectra also contain unimportant (non-characteristic) regions which add little (or no) weight to fitted values, e.g. energy regions in XRF spectra that contain little or no peak information.

Oxygen K-edge X-ray absorption spectra of liquids with minimization of window contamination.

Oxygen K-edge X-ray absorption spectroscopy is used routinely to study a range of solid materials. However, liquid samples are studied less frequently at the oxygen K-edge due to the combined challenges of high-vacuum conditions and oxygen contamination of window materials. A modular sample holder design with a twist-seal sample containment system that provides a simple method to encapsulate liquid samples under high-vacuum conditions is presented. This work shows that pure silicon nitride windows have lower oxygen contamination than both diamond- and silicon-rich nitride windows, that the levels of oxygen contamination are related to the age of the windows, and provides a protocol for minimizing the background oxygen contamination. Acid-washed 100 nm-thick silicon nitride windows were found to give good quality oxygen K-edge data on dilute liquid samples.

Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a zinc-dependent sulfur transfer protein.

The biosynthesis of Fe-S clusters in Bacillus subtilis and other Gram-positive bacteria is catalyzed by the SufCDSUB system. The first step in this pathway involves the sulfur mobilization from the free amino acid cysteine to a sulfur acceptor protein SufU via a PLP-dependent cysteine desulfurase SufS. In this reaction scheme, the formation of an enzyme S-covalent intermediate is followed by the binding of SufU. This event leads to the second half of the reaction where a deprotonated thiol of SufU promotes the nucleophilic attack onto the persulfide intermediate of SufS. Kinetic analysis combined with spectroscopic methods identified that the presence of a zinc atom tightly bound to SufU (Ka = 10(17) M(-1)) is crucial for its structural and catalytic competency. Fe-S cluster assembly experiments showed that despite the high degree of sequence and structural similarity to the ortholog enzyme IscU, the B. subtilis SufU does not act as a standard Fe-S cluster scaffold protein. The involvement of SufU as a dedicated agent of sulfur transfer, rather than as an assembly scaffold, in the biogenesis of Fe-S clusters in Gram-positive microbes indicates distinct strategies used by bacterial systems to assemble Fe-S clusters.

Structural characterization of CO-inhibited Mo-nitrogenase by combined application of nuclear resonance vibrational spectroscopy, extended X-ray absorption fine structure, and density functional theory: new insights into the effects of CO binding and the role of the interstitial atom.

The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm(-1) mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm(-1), additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by (13)CO isotope shifts). The EXAFS for wild-type N2ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal -CO and a partially reduced -CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational "shake" modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N2 reactivity of N2ase are discussed.

Facile electrocatalytic proton reduction by a [Fe-Fe]-hydrogenase bio-inspired synthetic model bearing a terminal CN- ligand.

An azadithiolate bridged CN- bound pentacarbonyl bis-iron complex, mimicking the active site of [Fe-Fe] H2ase is synthesized. The geometric and electronic structure of this complex is elucidated using a combination of EXAFS analysis, infrared and Mössbauer spectroscopy and DFT calculations. The electrochemical investigations show that complex 1 effectively reduces H+ to H2 between pH 0-3 at diffusion-controlled rates (1011 M-1 s-1) i.e. 108 s-1 at pH 3 with an overpotential of 140 mV. Electrochemical analysis and DFT calculations suggests that a CN- ligand increases the pKa of the cluster enabling hydrogen production from its Fe(i)-Fe(0) state at pHs much higher and overpotential much lower than its precursor bis-iron hexacarbonyl model which is active in its Fe(0)-Fe(0) state. The formation of a terminal Fe-H species, evidenced by spectroelectrochemistry in organic solvent, via a rate determining proton coupled electron transfer step and protonation of the adjacent azadithiolate, lowers the kinetic barrier leading to diffusion controlled rates of H2 evolution. The stereo-electronic factors enhance its catalytic rate by 3 order of magnitude relative to a bis-iron hexacarbonyl precursor at the same pH and potential.

Temperature-dependent iron motion in extremophile rubredoxins - no need for 'corresponding states'.

Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent.

Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron 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. 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 Fe-S 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)Fe-S cluster. Electron paramagnetic resonance (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 of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.

Characterization of [4Fe-4S] cluster vibrations and structure in nitrogenase Fe protein at three oxidation levels via combined NRVS, EXAFS, and DFT analyses.

Azotobacter vinelandii nitrogenase Fe protein (Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and 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.

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold.

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydF(EG)), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydF(EG). The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.

Redox, haem and CO in enzymatic catalysis and regulation.

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 Å (1 Å=0.1 nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.

Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm-1 and 1679 cm-1 together with positive bands at 1711 cm-1, 2135 and 2123 cm-1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm-1 is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol-1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm-1 with a positive 'Lo-2' band at 1780 cm-1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm-1 and a positive feature 'Lo-3' band at 1921 cm-1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

[FeFe]-hydrogenase maturation: HydG-catalyzed synthesis of carbon monoxide.

Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.

A star-shaped heteronuclear Cr(III)Mn(II)3 species and its precise electronic and magnetic structure: spin frustration studied by X-ray spectroscopic, magnetic, and theoretical methods.

Molecular magnets incorporate transition-metal ions with organic groups providing a bridge to mediate magnetic exchange interactions between the ions. Among them are star-shaped molecules in which antiferromagnetic couplings between the central and peripheral atoms are predominantly present. Those configurations lead to an appreciable spin moment in the nonfrustrated ground state. In spite of its topologically simple magnetic structure, the [Cr(III)Mn(II)(3) (PyA)(6)Cl(3)] (CrMn(3)) molecule, in which PyA represents the monoanion of syn-pyridine-2-aldoxime, exhibits nontrivial magnetic properties, which emerge from the combined action of single-ion anisotropy and frustration. In the present work, we elucidate the underlying electronic and magnetic properties of the heteronuclear, spin-frustrated CrMn(3) molecule by applying X-ray magnetic circular dichroism (XMCD), as well as magnetization measurements in high magnetic fields, density functional theory, and ligand-field multiplet calculations. Quantum-model calculations based on a Heisenberg Hamiltonian augmented with local anisotropic terms enable us not only to improve the accuracy of the exchange interactions but also to determine the dominant local anisotropies. A discussion of the various spin Hamiltonian parameters not only leads to a validation of our element selective transition metal L edge XMCD spin moments at a magnetic field of 5 T and a temperature of 5 K but also allows us to monitor an interesting effect of anisotropy and frustration of the manganese and chromium ions.

Feasibility of Laboratory-Based EXAFS Spectroscopy with Cryogenic Detectors.

Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy is a powerful technique that gives element-specific information about the structure of molecules. The development of a laboratory EXAFS spectrometer capable of measuring transmission spectra would be a significant advance as the technique is currently only available at synchrotron radiation lightsources. Here, we explore the potential of cryogenic detectors as the energy resolving component of a laboratory transmission EXAFS instrument. We examine the energy resolution, count-rate, and detector stability needed for good EXAFS spectra and compare these to the properties of cryogenic detectors and conventional X-ray optics. We find that superconducting tunnel junction (STJ) detectors are well-suited for this application.

X-ray absorption spectroscopy of organic sulfoxides.

Organic sulfoxides, a group of compounds containing the sulfinyl S[double bond, length as m-dash]O group, are widespread in nature, important in health and disease, and used in a variety of applications in the pharmaceutical industry. We have examined the sulfur K-edge X-ray absorption near-edge spectra of a range of different sulfoxides and find that their spectra are remarkably similar. Spectra show an intense absorption peak that is comprised of two transitions; a S 1s → (S-O)σ* and a S 1s → [(S-O)π* + (S-C)σ*] transition. In most cases these are sufficiently close in energy that they are not properly resolved; however for dimethylsulfoxide the separation between these transitions increases in aqueous solution due to hydrogen bonding to the sulfinyl oxygen. We also examined tetrahydrothiophene sulfoxide using both the sulfur and oxygen K-edge. This compound has a mild degree of ring strain at the sulfur atom, which changes the energies of the two transitions so that the S 1s → [(S-O)π* + (S-C)σ*] is below the S 1s → (S-O)σ*. A comparison of the oxygen K-edge X-ray absorption near-edge spectra of tetrahydrothiophene sulfoxide with that of an unhindered sulfoxide shows little change, indicating that the electronic environment of oxygen is very similar.

Molybdenum trafficking for nitrogen fixation.

The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of nitrogen and carbon. Here we describe the trafficking of molybdenum for nitrogen fixation in the model diazotrophic bacterium Azotobacter vinelandii. The genes and proteins involved in molybdenum uptake, homeostasis, storage, regulation, and nitrogenase cofactor biosynthesis are reviewed. Molybdenum biochemistry in A. vinelandii reveals unexpected mechanisms and a new role for iron-sulfur clusters in the sequestration and delivery of molybdenum.

Extended X-ray absorption fine structure and nuclear resonance vibrational spectroscopy reveal that NifB-co, a FeMo-co precursor, comprises a 6Fe core with an interstitial light atom.

NifB-co, an Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway to the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy together with (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. The spectra have been interpreted in part by comparison with data for the completed FeMo-co attached to the NafY carrier protein: the NafY:FeMo-co complex. EXAFS analysis of the NifX:NifB-co complex yields an average Fe-S distance of 2.26 A and average Fe-Fe distances of 2.66 and 3.74 A. Search profile analyses reveal the presence of a single Fe-X (X = C, N, or O) interaction at 2.04 A, compared to a 2.00 A Fe-X interaction found in the NafY:FeMo-co EXAFS. This suggests that the interstitial light atom (X) proposed to be present in FeMo-co has already inserted at the NifB-co stage of biosynthesis. The NRVS exhibits strong bands from Fe-S stretching modes peaking around 270, 315, 385, and 408 cm(-1). Additional intensity at approximately 185-200 cm(-1) is interpreted as a set of cluster "breathing" modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data for NifX:NifB-co are best simulated using a Fe 6S 9X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. The results are consistent with the conclusion that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.