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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.

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.

Direct reactions of the MRI contrast agent K2[Gd(DTPA)(H2O)]·5H2O (1) (H5DTPA = diethylenetriaminepentaacetic acid) with dipotassium hydrogen phosphate (K2HPO4) or phosphite (K2HPO3) result in the isolation of well-defined Gd-DTPA phosphite K6[Gd2(DTPA)2(HPO3)]·7H2O (2) or phosphate K6[Gd2(DTPA)2(HPO4)]·10H2O (3), respectively. Their lanthanum analogs K4[La2(DTPA)2(H2O)]·8H2O (4), K6[La2(DTPA)2(HPO3)]·7H2O (5) and K6[La2(DTPA)2(HPO4)]·10H2O (6) are used for comparison. The phosphate and phosphite groups are able to substitute the coordinated water molecules in 1 and 4 in a close physiological aqueous solution, and act as bridging ligands to link adjacent Ln(DTPA)(2-) (Ln = Gd and La) into dimeric structures. Solid state and solution (13)C NMR spectra of dimer 4 show complete dissociation into its monomeric species in solution, while no dissociation is observed for lanthanum phosphite 5 and phosphate 6 in solution, which show only one set of (13)C spectra with the largest downfield shifts at 182.0 and 182.3 ppm respectively. Comparisons of the bond distances and spectral data indicate that the interaction between DTPA and central Ln(3+) cations are weakened after the substitutions, which support phosphate substituted Gd-DTPA as an initial intermediate in nephrogenic systemic fibrosis.

Mo and Fe K-edge EXAFS analysis of NifQ shows the presence of a [MoFe3S4] cluster and a second independent Mo environment that includes Mo-O bonds and Mo-S bonds. Both environments are relevant to FeMo-co biosynthesis and may represent different stages of Mo biochemical transformations catalyzed by NifQ.

Piecing Together Hydrogenase Microbial hydrogenase enzymes generally use iron to catalyze the reversible formation of hydrogen from protons and electrons. Key to their efficiency is a set of iron-coordinating ligands, including CO and cyanide. Kuchenreuther et al. (p. 472 ) examined how the HydG maturase enzyme breaks down the amino acid tyrosine to derive these diatomic ligands for assembly of the diiron class of hydrogenases. The first step involves abstraction of an H atom from the phenolic OH substituent of the side chain. Electron paramagnetic resonance spectroscopy revealed a radical intermediate that subsequently results from heterolysis of the bond tethering the side chain to the α-carbon. With the side chain thus jettisoned, the residual dehydroglycine could be transformed into CO and CN − .

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.

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.

A ferredoxin isolated from the archaeon Sulfolobus acidocaldarius strain DSM 639 has been shown to contain one [3Fe-4S]1 + 10 cluster with a reduction potential of -275 mV and one [4Fe-4S]2+/1+ cluster with a reduction potential of -529 mV at pH 6.4, in the temperature range 0-50 degrees C. The monomer molecular mass was confirmed to be 10907.5 +/- 1.0 Da by electrospray mass spectrometry, as calculated from the published amino acid sequence [Minami, Y. Wakabayashi. S., Wada, K., Matsubara, H., Kerscher, L. and Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751], while the holoprotein molecular mass was found to be 11,550 +/- 1.0 Da. The reduced [3Fe-4S]0 cluster was also shown by direct electrochemistry and magnetic circular dichroic spectroscopy to undergo a one-proton uptake reaction as first observed for Azotobacter chroococcum ferredoxin I [George, S. J., Richards, A. J. M., Thomson, A. J. and Yates, M. G. (1984) Biochem. J. 224, 247-251]. The pKa of the protonation step has been determined by a novel thin film electrochemical method to be 5.8. This is significantly different from the pKa of 7.7 determined for A. vinelandii ferredoxin I [Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, J. M., Stephens, P. J., LaMar, G. N., Gorst, C. M. and Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939] and indicates that the polypeptide chain around the [3Fe-4S] cluster controls this reaction. Although this appears to be only the second reported case of protonation at or near the reduced [3Fe-4S]0 cluster, its observation in S. acidocaldarius ferredoxin raises the question of the generality of this chemistry for 3Fe clusters. The similarity of the pKa to the estimated intracellular pH of S. acidocaldarius strongly suggests a physiological role for this process.

Fast diffusion-dominated electron transfer between Azotobacter chroococcum 7Fe ferredoxin, FdI, and pyrolytic graphite ‘edge’ electrodes, promoted by aminoglycosides, permits detailed voltammetric studies and preparation of oxidation states inaccessible by chemical titration. The [3Fe-4S] cluster exhibits pH dependent E 1 2 values (30°C); E 1 2 (alkaline) = 460 ± 10 mV vs NHE, −d E 1 2 /d(pH) = 55 mV, p K = 7.8. The [4Fe-4S] cluster is characterised by an unusually low reduction potential, −645 ± 10 mV vs NHE, at pH 8.3, with a slight pH dependence, −d E 1 2 /d(pH) ∼25 mV over the pH range 8.5-7.0. No redox couple is observed at potentials between −300 and +600 mV vs NHE. This shows that the [4Fe-4S] cluster is not an HIPIP-type centre. The electron paramagnetic resonance spectrum, centred at g = 1.93, of the product resulting from bulk electrolysis at −835 mV is assigned to a [4Fe-4S] + cluster interacting magnetically with a reduced [3Fe-4S] cluster.

We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Iron-sulfur protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the crystal structure.

Summary Background Nephrogenic systemic fibrosis (NSF) is an incurable, debilitating disease found exclusively in patients with decreased kidney function and comprises a fibrosing disorder of the skin and systemic tissues. The disease is associated with exposure to gadolinium (Gd)-based contrast agents (GBCA) used in magnetic resonance imaging (MRI). Tissue samples from many patients with NSF contain micron-sized insoluble Gd-containing deposits. However, the precise composition and chemical nature of these particles is unclear. Objectives To clarify the precise chemical structure of the Gd-containing deposits in NSF tissues. Methods Autopsy skin tissues from a patient with NSF were examined in situ using synchrotron X-ray fluorescence (SXRF) microscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy and in correlation with light microscopy and the results of scanning electron microscopy/energy dispersive spectroscopy analyses. Results The insoluble Gd deposits were shown to contain Gd no longer coordinated by GBCA chelator molecules but rather in a sodium calcium phosphate material. SXRF microscopy shows a clear correlation between Gd, Ca and P. EXAFS spectroscopy shows a very different spectrum from the GBCAs, with Gd–P distances at 3·11 A and 3·72 A as well as Gd–Gd distances at an average of 4·05 A, consistent with a GdPO4 structure. Conclusions This is the first direct evidence for the chemical release of Gd from GBCA in human tissue. This supports the physical–chemical, clinical and epidemiological data indicating a link between stability and dose of GBCA to the development of NSF.

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.

Under intense soft X-ray irradiation, we have observed time-dependent changes in the soft X-ray spectra of virtually all the Fe coordination complexes that we have examined, indicating chemical transformation of the compound under study. Each compound, with oxidation states ranging from Fe(IV) to Fe(0), has been studied with either Fe L-edge spectroscopy or N K-edge spectroscopy. We find that very often a well-defined spectroscopic change occurs, at least initially, which is apparently capable of straightforward interpretation in terms of X-ray induced photoreduction, photooxidation or ligand photolysis. We briefly discuss the probable chemical nature of the changes and then estimate the rate of chemical change, thereby establishing the necessary radiation dose. We also demonstrate that the photochemistry not only depends on the Fe oxidation state but also the coordination chemistry of the complex. It seems that a proper understanding of such X-ray photochemical effects could well greatly assist the assignment of soft X-ray spectra of uncharacterized metal sites.

Cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d1 dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr25 dissociates from the distal side of heme d1, and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd1 is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d1 is EPR-silent. Magnetic circular dichroism studies indicate that heme d1 is low-spin FeIII but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at FeIII heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd1 from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent FeIII heme d1 species in nitrite reduction.

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.

Nitrogenase catalyzes a reaction critical for life, the reduction of N(2) to 2NH(3), yet we still know relatively little about its catalytic mechanism. We have used the synchrotron technique of (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the dynamics of the Fe-S clusters in this enzyme. The catalytic site FeMo-cofactor exhibits a strong signal near 190 cm(-)(1), where conventional Fe-S clusters have weak NRVS. This intensity is ascribed to cluster breathing modes whose frequency is raised by an interstitial atom. A variety of Fe-S stretching modes are also observed between 250 and 400 cm(-)(1). This work is the first spectroscopic information about the vibrational modes of the intact nitrogenase FeMo-cofactor and P-cluster.

X-ray magnetic circular dichroism (XMCD) spectroscopy is a powerful emerging technique that measures difference in absorption of left- and right-circularly polarized X-rays by a magnetized sample, often at cryogenic temperatures. It is already well established in magnetic materials science, and it is likely to become a significant tool for the inorganic and bioinorganic communities. As with all X-ray spectroscopies, XMCD has the advantage of being element specific. Interpretation of the spectra can: provide quantitative information about the distribution of spin and orbital angular momenta from simple “sum rules”; determine spin orientations from the sign of the XMCD signal; infer spin states from magnetization curves; and separate magnetic and non-magnetic components in heterogeneous samples. With new synchrotron radiation sources and improved end stations, XMCD measurements on dilute samples such as metals in enzymes, are becoming more routine. This review first details the technology currently available for XMCD measurements and outlines the theory underlying interpretation of the spectra. It then illustrates the strengths of the XMCD technique using examples taken from bioinorganic chemistry and materials science. In this way, we aim to encourage chemists, materials scientists, and biologists to consider XMCD spectroscopy as an approach to understanding the electronic and magnetic structure of their samples.

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium vinosum can exist in three different redox states. In the most oxidized state (Ni(a)-S) the nickel is divalent. The most reduced state (Ni(a)-SR) likewise has Ni(2+), while the intermediate state (Ni(a)-C) has Ni(3+). The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred from the data that the Ni(a)-S --Ni(a)-C* and Ni(a)-C* --Ni(a)-SR transitions induced by dihydrogen require one of the [4Fe-4S] clusters to be oxidized. Enzyme in the Ni(a)-S* state with all of the iron-sulfur clusters reduced reacts with dihydrogen to form the Ni(a)-SR state in milliseconds. By contrast, when one of the cubane clusters is oxidized, the Ni(a)-S state reacts with dihydrogen to form the Ni(a)-C state with all of the iron-sulfur clusters reduced. The competition between dihydrogen and carbon monoxide for binding to the active site was dependent on the redox state of the nickel ion. Formation of the Ni(a)-S.CO state (Ni(2+)) by reacting CO with enzyme in the Ni(a)-SR and Ni(a)-S states (Ni(2+)) is considerably faster than its formation from enzyme in the Ni(a)-C* (Ni(3+)) state. Excess oxygen converted hydrogen-reduced enzyme to the inactive Ni(r)* state within 158 ms, suggesting a direct reaction at the Ni-Fe site. With lower O(2) concentrations the formation of intermediate states was observed. The results are discussed in the light of the present knowledge of the structure and mechanism of action of the A. vinosum enzyme.

The reaction between hydrogen and the [NiFe]-hydrogenase from Allochromatium vinosum in its inactive form has been studied by stopped-flow infrared spectroscopy. The data, for the first time, clearly show that at room temperature enzyme in the unready state, either oxidized or reduced, does not react with hydrogen. Enzyme in the ready state reacts with hydrogen after a lag phase of about six seconds, whereby a specific reduction of the enzyme occurs. The lag phase and the rate of reduction of the ready enzyme are neither dependent on the enzyme concentration nor on the substrate concentration, i.e., substoichiometric and 8-fold excess amounts of H(2) reduce the ready enzyme at the same rate. Oxygen delays this reaction but does not prevent it. The infrared changes lead us to suggest that the hydroxyl group, bridging between the Ni and the Fe atom in the active site, becomes protonated during this reduction. At physiological temperatures, this property of the inactive ready enzyme enables a full development of activity by substoichiometric H(2) concentrations.

This paper describes the kinetics and intimate mechanisms associated with cyanation of [2Fe3S] assemblies to give species structurally related to the subsite of all-iron hydrogenase. Stopped-flow FTIR spectroscopy has enabled the quantitation of the dynamics of five well-defined steps that experimentally illustrate the role of bridging carbonyls in the assembly of the dicyanide species, how on-off sulfur ligation can have a dramatic effect on cyanation kinetics and how the [2Fe3S] core stabilises bridging carbonyl species.

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 A 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.

Three iron-sulfur proteins–HydE, HydF, and HydG–play a key role in the synthesis of the [2Fe] H component of the catalytic H-cluster of FeFe hydrogenase. The radical S -adenosyl- l -methionine enzyme HydG lyses free tyrosine to produce p -cresol and the CO and CN − ligands of the [2Fe] H cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN − ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the 13 C, 15 N, and 57 Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO) 2 (CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe] H component of the H-cluster.

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.

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.

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g = 2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl2 results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl2 at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca2+ do not result in restoration of activity. Experiments with 45Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca2+ ions in LiP H2. It is concluded that both the structural Ca2+ ions play key roles in the reversible alkaline inactivation of LiP H2.

We have developed a general theoretical approach for analyzing the intensities of magnetic circular dichroism (MCD) spectra of paramagnetic species with S>1/2 in the nonlinear regions of temperature and magnetic field. The method takes full advantage of the irreducible tensor method in order to obtain maximum simplification from symmetry. The approach, which is based on a detailed treatment of spin-orbit coupling and Zeeman interaction in terms of the symmetry properties of basis sets of wave functions, factorizes contributions into bands with Gaussian and derivative shapes in order to extend earlier treatments based on the so-called linear field limit. The method is applied to analyze and fit the form of the MCD spectra and the MCD magnetization curves of pseudo-tetrahedral high-spin Fe(III), S=5/2, in the protein rubredoxin from Desulfovibrio gigas, a representative of a family of iron–sulphur proteins. This treatment provides for the first time a satisfactory fit of these curves over a temperature rang...

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pKas at 7.4 and 4.0. Data obtained with the His-42 → Leu [(H42L)HRPC*)] and Arg-38 → Leu [(R38L)HRPC*] mutants allowed the pKa at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν (CO) bands at 1934 cm–1 and 1905 cm–1 whose relative intensity changed with pH, showing an acidic and a basic pKa as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118 : 3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2 cm–1 (pH 7.0) and 1941.5 cm–1 (pH 5.0) respectively. Acidic and alkaline pKas were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Soret maxima. (H42L)HRPC*-CO exhibited a pKa at ∼pH 4.0 but no alkaline pKa. (R38L)HRPC*-CO exhibited a single pKa at pH 6.5. Shifts of 2–3 cm–1 in ν (CO) with (H42L)HRPC*-CO in D2O show that a distal residue is H-bonding to the CO in this variant at both pD 7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν (CO) band was observed at pD 5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH 8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pKa between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.

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.

Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5–5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108000 to 148000±2000 M-1·cm-1 (results given ±S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.

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., National Institutes of Health (U.S.) (NIH grant GM69857), National Institutes of Health (U.S.) (NIH grant GM39451), National Institutes of Health (U.S.) (NIH grant HL 102662), National Institutes of Health (U.S.) (NIH grant GM65440), National Institutes of Health (U.S.) (NIH grant GM48242), National Institutes of Health (U.S.) (NIH grant Y1-GM- 1104), National Institutes of Health (U.S.) (NIH grant GM065318), National Institutes of Health (U.S.) (NIH grant AG027349), National Science Foundation (U.S.) (grant number CHE-0745353), United States. Dept. of Energy. Office of Biological and Environmental Research, Howard Hughes Medical Institute (Investigator)

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 A on PA binding with the apparent appearance of a new Fe-Fe interaction at 3.99 A. An analogous change in the long Mo-Fe 5.1 A 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 A. This study comprises the first experimental evidence of the conformational changes of the FeMo-cofactor active site on binding a substrate or product.

Extensive nitrogen K-edge X-ray absorption studies have been performed for the first time on seven petroleum asphaltenes and nitrogen standard compounds for the purpose of determining chemical forms of nitrogen present in the asphaltenes; such an objective is difficult to achieve by any other method. Sulfur XANES studies on fossil fuel samples have provided a rich source of information for the last ten years; similar XANES studies of nitrogen in fossil fuels have only recently been successfully performed using new, advanced fluorescence detection. Generally, the spectra of different chemical forms of nitrogen produce readily distinguishable features, thereby facilitating asphaltene analysis. Approximate contributions from different nitrogen structures present in the asphaltenes are calculated by comparing normalized areas under corresponding resonances in the spectra of the asphaltenes and the model compounds. The standard model compounds of nitrogen studied are pyrroles, pyridines, saturated amines, and metalloporphyrins. Most of the nitrogen in asphaltenes is found to be present in aromatic forms, with a very small amount as saturated amine. The pyrrole form of nitrogen is more abundant than the pyridine form in asphaltenes, and the pyridine fraction in different asphaltenes is somewhat variable. Pyridine, which is more basic than pyrrole, shows [pi][sup *] resonancesmore » shifted to significantly lower energies than those observed for pyrroles. The relative positions of nitrogen [pi][sup *] resonances are determined according to whether the nitrogen lone pair of electrons is shared in the [pi] aromatic system. These spectral shifts are largely produced by resonance effects rather than inductive effects, which are observed in the sulfur case. Spectra of more complicated molecules such as porphyrins and imidazoles are explained along similar lines. The saturated amine shows only a [sigma][sup *] resonance. 6 figs., 1 tab.« less

Here, we report the impact of substitution for the α-70Val residue of Azotobacter vinelandii nitrogenase molybdenum-iron (MoFe) protein on the so-called “hi-CO” CO bound complex as monitored in real-time by stopped-flow infra-red (SF-IR) spectroscopy. Nitrogenase is a bacterial metalloenzyme system whose physiological function is to catalyze the reduction of dinitrogen to ammonia [1, 2] with a concomitant reduction of 2H+ to H2 and hydrolysis of MgATP. X-ray crystallography on MoFe nitrogenase reveals the active-site FeMo-cofactor to be an unprecedented [Fe7S9MoX:homocitrate] cluster [3, 4], figure 1. However, simple inspection of this structure does not provide an obvious location for substrate binding or any indication of the subsequent mechanism for substrate reduction. Mechanisms focused on substrate binding to either Mo and Fe sites have been proposed as have combination approaches that involve migration of substrate-derived moieties between metal atoms during reduction [5–8].

BACKGROUND: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. PRINCIPAL FINDINGS: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms. SIGNIFICANCE: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

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.

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 [CrIIIMnII3(PyA)6Cl3] (CrMn3) 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 CrMn3 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.

Fluorescence detection methods have been developed for measuring the L 2.3 X-ray absorption spectra of first transition series metalloprotiens. Samples are prepared as thin films on silicon supports, and mounted on a liquid helium cooled cold finger in a UHV chamber. A windowless Ge array detector discriminates metal L fluorescence from oxygen K a background. The high resolution, strong sensitivity to chemical environment and amenability to quantitative spectral shape analysis indicate that L-edges of the first transition series metals are a useful probe for bioinorganic studies.

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.

Cytochrome c″ (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.

Fast diffusion-dominated electron transfer between bacterial ferredoxins and pyrolytic graphite 'edge' electrodes promoted by aminoglycosides permits detailed voltammetric studies and preparation of oxidation states inacessible by chemical titration. Low temperature EPR and magnetic circular dichroism spectroscopy identifies the cluster types, and magnetic states at defined oxidation levels. In the 7Fe ferredoxin, Fd 11, Azotobacter &roococcum, the (3Fe-4S) cluster exhibits pH dependent El/ values (3OOC): El 2 (alkaline) = -460 f 10 mV vs NHE, -dE1/2/d(pH) = 55 mV, pK = 5.8. The (4Fe-4S) duster is characterised by an unusually low reduction potential, -645f 10 mV vs NHE at pH 8.3. FdIII, PesulphovibriQj&icanyS, contains one (3Fe-4S), and one (4Fe-4S) cluster when isolated aerobically. Cyclic voltammetry shows that w reduced the (3Fe-4SIo centre reacts rapidly with ferrous ion to form a (4Fe-4S) cluster. Since the protein sequence contains only seven cysteines this cluster must possess non-cysteinyl ligation, which we propose to be the carboxylate side-chain of aspartic acid, residue 14. The magnetic properties of this cluster in the reduced state are S = 3/2. 9

Cyclic voltammetry readily visualizes the redox properties of many proteins. Net electron exchange between the protein and an electrode produces an electrical current that simultaneously quantitates and characterizes the underlying redox event(s). However, no direct information regarding the molecular origin, or consequences, of electron transfer is available. Integrating voltammetric and spectroscopic methods is one route to a more 'holistic' description of protein electron transfer. Here, we illustrate this approach with spectroelectrochemical studies of Rhodovulum sulfidophilum cytochrome c 2 and Escherichia coli cytochrome bd that employ electronic absorbance, infra-red and magnetic circular dichroism spectroscopies. © 2007 Springer-Verlag.

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S(cys))(4) site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe-S stretching modes are observed between 355 and 375 cm(-1); upon reduction these modes shift to 300-320 cm(-1). This is the first observation of Fe-S stretching modes in a reduced Rd. The peak in S-Fe-S bend mode intensity is at approximately 150 cm(-1) for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm(-1) for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The (57)Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey-Bradley force fields. The three main bands were qualitatively reproduced using a D(2)(d) Fe(SC)(4) model. A C(1) Fe(SCC)(4) model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe-S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant K(Fe-S) for Fe-S stretching modes ranged from 1.24 to 1.32 mdyn/A. K(Fe-S) is clearly diminished in reduced Rd; values from approximately 0.89 to 1.00 mdyn/A were derived from different models. In contrast, in the final models the force constants for S-Fe-S bending motion, H(S-Fe-S), were 0.18 mdyn/A for oxidized Rd and 0.15 mdyn/A for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.

[NEt(4)][FeCl(4)], [P(C(6)H(5))(4)][FeCl(4)], and [NEt(4)](2)[Fe(2)S(2)Cl(4)] have been examined using (57)Fe nuclear resonance vibrational spectroscopy (NRVS). These complexes serve as simple models for Fe-S clusters in metalloproteins. The (57)Fe partial vibrational density of states (PVDOS) spectra were interpreted by computation of the normal modes assuming Urey-Bradley force fields, using additional information from infrared and Raman spectra. Previously published force constants were used as initial values; the new constraints from NRVS frequencies and amplitudes were then used to refine the force field parameters in a nonlinear least-squares analysis. The normal-mode calculations were able to quantitatively reproduce both the frequencies and the amplitudes of the intramolecular-mode (57)Fe PVDOS. The optimized force constants for bending, stretching, and nonbonded interactions agree well with previously reported values. In addition, the NRVS technique also allowed clear observation of anion-cation lattice modes below 100 cm(-1) that are nontrivial to observe by conventional spectroscopies. These features were successfully reproduced, either by assuming whole-body motions of point-mass anions and cations or by simulations using all of the atoms in the unit cell. The advantages of a combined NRVS, Raman, and IR approach to characterization of Fe-S complexes are discussed.

High-resolution molybdenum K-edge X-ray emission spectroscopy (XES) was used to characterize the K beta(4) and K beta' ' valence-to-core transition bands in the oxo-Mo compounds K(2)MoO(4), MoO(S(2)CNEt(2))(2), and MoO(2)(S(2)CNEt(2))(2). The K beta(4) and K beta' ' emission bands are attributed to transitions to the Mo 1s core hole from molecular orbitals possessing primarily molybdenum 4d and oxygen 2s character, respectively. This communication describes the first assignment of the K beta' ' interatomic band in the emission spectra of molybdenum complexes. Additionally, the K beta(4) and K beta' 'transitions are shown to be sensitive to the chemical and electronic environment of the metal, suggesting that high-resolution XES might be an effective method for elucidating the nature of the molybdenum centers in biological systems.

Iron(I) in biology?: one-electron oxidation of an {Fe(I)–Fe(I)} carbonyl cyanide precursor bearing a proximal thioether group leads to an {Fe(I)–Fe(II)} bridging carbonyl transient with spectral features similar to the di-iron sub-site of the CO inhibited paramagnetic centre of all-iron hydrogenase.

The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.4 +/- 0.5) x 10(4) M(-1) x s(-1), which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket. Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm(-1), suggesting a distorted Fe-N-O coordination geometry. The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k(6-5) = (8.1 +/- 0.7) x 10(3) M(-1) x s(-1), implying that an additional molecule of NO is required to form the 5c-NO adduct. Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c' binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position. The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c' and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts.