Stephen P. Cramer

A 13-element Ge detector for fluorescence EXAFS

Abstract At low concentrations, recording X-ray absorption spectra in fluorescence excitation mode is more sensitive than transmission mode. For dilute samples, the fluorescence signal is often obscured by scattered X-rays, and matrix and filter fluorescence. To discriminate against this background, while maintaining a large angular acceptance and high count rate capability, we have constructed a new detection system based on an array of intrinsic Ge detectors. The device uses 13 individual 11 mm diameter Ge detectors, clustered in a 1:3:5:3:1 pattern on a common cryostat, combined with Soller slits and filters to reduce the background signals. Pulsed optical feedback preamplifiers are followed by Gaussian-shaping amplifiers having fast discriminators to register the incoming count rate (ICR). Correction for dead time using the ICR signal allowed operation in the vicinity of 75 kHz per channel, with a 1 μs shaping time at 6 keV. For lower count rate applications, an average resolution of 160 eV at 5.9 keV was obtained with 8 μs shaping. Recent experience with this detector at the Stanford Synchrotron Radiation Laboratory is presented. The performance is illustrated using spectra obtained from phosphorus compounds and a thin iridium foil. The performance of this device is compared with previous fluorescence detection schemes, such as those using filter/slit combinations or barrel monochromators.

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 (HydFEG), 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 HydFEG. 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.

A systematic x-ray absorption study of molybdenum complexes. The accuracy of structural information from extended x-ray absorption fine structure

X-ray absorption spectra have been collected using synchrotron radiation for a number of mononuclear and dinuclear molybdenum complexes containing carbon, nitrogen, oxygen, and sulfur donor atoms. The extended fine structure (EXAFS) of the Mo absorption edge has been analyzed by a method which combines Fourier transform and curve-fitting techniques. Parameterized phase shift and amplitude functions for describing Mo-C, Mo-N, Mo-0, Mo-S, and Mo-Mo interactions were obtained from spectra of Mo(CO)6, M o ( N C S ) ~ ~ , MOO^^-, M o ( S ~ C ~ H ~ ) ~ , and M0204cys2~-. Application of these phase shifts and amplitudes to the EXAFS of other compounds yielded distance determinations to an accuracy consistently better than 0.03 A for atoms bound to Mo. The number and type of coordinating atoms were also determined with a reasonable degree of certainty. This work demonstrates the applicability (and limitations) of EXAFS for providing structural information about a specific absorbing center under noncrystalline conditions, and it lays a foundation for the analysis of the x-ray absorption spectra of nitrogenase and other Mo proteins.

(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.

High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Background The realization of hydrogenase-based technologies for renewable H2 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 H2 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 H2-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.

Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center

Reactions of nitric oxide with cysteine-ligated iron-sulfur cluster proteins typically result in disassembly of the iron-sulfur core and formation of dinitrosyl iron complexes (DNICs). Here we report the first evidence that DNICs also form in the reaction of NO with Rieske-type [2Fe-2S] clusters. Upon treatment of a Rieske protein, component C of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1, with an excess of NO(g) or NO-generators S-nitroso-N-acetyl-D,L-pencillamine and diethylamine NONOate, the absorbance bands of the [2Fe-2S] cluster are extinguished and replaced by a new feature that slowly grows in at 367 nm. Analysis of the reaction products by electron paramagnetic resonance, Mössbauer, and nuclear resonance vibrational spectroscopy reveals that the primary product of the reaction is a thiolate-bridged diiron tetranitrosyl species, [Fe(2)(μ-SCys)(2)(NO)(4)], having a Roussins red ester (RRE) formula, and that mononuclear DNICs account for only a minor fraction of nitrosylated iron. Reduction of this RRE reaction product with sodium dithionite produces the one-electron-reduced RRE, having absorptions at 640 and 960 nm. These results demonstrate that NO reacts readily with a Rieske center in a protein and suggest that dinuclear RRE species, not mononuclear DNICs, may be the primary iron dinitrosyl species responsible for the pathological and physiological effects of nitric oxide in such systems in biology.

Bulk-sensitive XAS characterization of light elements: from X-ray Raman scattering to X-ray Raman spectroscopy

X-Ray absorption spectroscopy (XAS) is a powerful tool for element-specific characterization of local structure and chemistry. Although application of XAS in the hard X-ray region is now routine, the soft X-ray region (containing light-element K-edges) presents a number of experimental problems. Most of the difficulties, including surface sensitivity, restricted sample environments, and radiation damage, stem from the submicron path lengths of soft X-rays and/or electrons. X-Ray Raman scattering (XRS) provides a means for obtaining the information content of soft X-ray spectra while maintaining the experimental benefits of hard X-ray techniques. In the XRS process, an incident photon is inelastically scattered and part of its energy is transferred to excite an inner shell electron into an unoccupied state. Under the dipole approximation, the resulting features are identical to the corresponding XAS spectrum. In the past, the extremely low cross-section of XRS has made this technique impractical, but intense new X-ray facilities and improvements in X-ray optics have helped to put XRS on the brink of becoming a routine spectroscopic tool. At present, high-quality X-ray Raman spectra can be obtained in minutes to hours. X-Ray Raman spectroscopy thus represents a hard X-ray alternative to conventional XAS techniques in the study of systems with light elements, including C, N and O. In this review we describe the technique, present examples of recent work, and discuss the prospects for the future.

Amorphous MoS3 and WS3

Abstract MoS 3 and WS 3 are examples of a group of transition metal chalcogenides which have only been prepared in amorphous form. Recently these compounds have been found to show interesting properties as cathode materials in ambient temperature alkali metal batteries. In particular, MoS 3 reacts readily and reversibly with lithium, giving a battery system with a high theoretical energy density. In this paper, we report results of structural studies of amorphous MoS 3 and WS 3 using X-ray radial distribution analysis, EXAFS, X-ray photoelectron spectroscopy, magnetic susceptibility measurements and vibrational spectroscopies. The results reveal that these amorphous compounds have a chain-like structure similar to that of the crystalline trichalcogenides of the neighboring IVB and VB elements. However, the combined formations of the metal dimers and polysulfide bonds along the chains are structural features unique to these amorphous compounds. The possible implications of this structure to the observed electrochemical behavior will be discussed.

In situ x-ray absorption spectroscopic study of the Li[Ni1/3Co1/3Mn1/3]O2 cathode material

The layered LiNi1∕3Co1∕3Mn1∕3O2 system has recently drawn considerable interest for use as a cathode material for rechargeable lithium batteries. In order to investigate the charge-compensation mechanism and structural perturbations occurring in the system during cycling, in situ x-ray absorption spectroscopy (XAS) measurements were performed utilizing a novel electrochemical in situ cell specifically designed for long term x-ray experiments. The cell was cycled at a moderate rate through a typical Li-ion battery operating voltage range (2.9–4.7 V). The electrode contained 2.025 mg of LiNi1∕3Co1∕3Mn1∕3O2 on a 25-μm Al foil and had an area of 0.79cm2. The x-ray absorption spectroscopy (XAS) measurements were performed at the Ni, Co, and the Mn edges at different states of charge (SOC) during cycling, revealing details about the response of the cathode to Li insertion and extraction processes. Changes of bond distance and coordination number of Ni, Co, and Mn absorbers as a function of the state of charge o...

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

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 13C, 15N, and 57Fe 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. Vibrational spectroscopy traces the origin of carbon monoxide and cyanide ligands in the active site of di-iron hydrogenase enzymes. [Also see Perspective by Pickett] Sourcing CO and Cyanide Hydrogenase enzymes derive their activity in part from the coordination of CO and cyanide ligands to metals in their active sites. Recent work elucidated the jettisoning of a tyrosine side chain at the outset of the biosynthetic pathway toward these ligands in the di-iron class of hydrogenase. Kuchenreuther et al. (p. 424; see the Perspective by Pickett) now apply stopped-flow infrared spectroscopy to uncover the next portion of the pathway, during which the residual tyrosine fragment is further broken down into CO and CN− ligands at a single iron center in an iron sulfur cluster associated with the HydG enzyme.