S K- and Mo L-edge X-ray absorption spectroscopy to determine metal-ligand charge distribution in molybdenum-sulfur compounds.

Mo L-edge and S K-edge X-ray absorption spectroscopy were applied to investigate the charge distribution between Mo and S in a series of Mo thiolate compounds, which serve as amide-sulfur H-bonding models and exhibit different redox potentials arising from polar group effects and ligand hydrogen bonds near the redox center. For all oxidized complexes, the S K-edge spectra exhibit a thiolate-based pre-edge feature centered at 2470.2 eV and the inflection point oCCurs at 2472.0 eV. No intense pre-edge feature is observed in the spectra for the reduced Mo model compounds and the energy shift of the S K-edge position depends on the S-ligand. Correlations between ligand charge density and the redox potential of the Mo-S cores are observed.

Absence of Mn-centered oxidation in the S(2) --> S(3) transition: implications for the mechanism of photosynthetic water oxidation.

A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) --> [S(4)] --> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) --> S(3) transition, in contrast to the S(0) --> S(1) and S(1) --> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) --> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.

Counting the number of disulfides and thiol groups in proteins and a novel approach for determining the local pKa for cysteine groups in proteins in vivo.

X-ray Absorption Spectroscopy (XAS) is a powerful tool to investigate sulfur in biological molecules. The spectral features are sensitive to the local electronic and geometric environment of the atom; thus, they constitute a fingerprint of the different chemical forms in which the sulfur is present. This allows straightforward detection of the ratio between free thiols and disulfides. Intra- or inter-molecular disulfide bond formation between residues plays an important role in structural and conformational changes in proteins, and such changes can be investigated using sulfur XAS. Also, a thiolate-disulfide equilibrium is involved in the regulation of the redox potential in the cells by means of modulating the concentrations of the reduced (thiolate) and oxidized (disulfide) form of the tripeptide glutathione. Thus, we can monitor the redox state of a cell by means of sulfur XAS. Thiols also exhibit an acid-base equilibrium, and sulfur XAS can be used to determine the local pKa of the -SH group. Here we report examples of how sulfur XAS has been used for these applications.

X-ray spectroscopy-based structure of the Mn cluster and mechanism of photosynthetic oxygen evolution.

The mechanism by which the Mn-containing oxygen evolving complex (OEC) produces oxygen from water has been of great interest for over 40 years. This review focuses on how X-ray spectroscopy has provided important information about the structure of this Mn complex and its intermediates, or S-states, in the water oxidation cycle. X-ray absorption near-edge structure spectroscopy and high-resolution Mn Kbeta X-ray emission spectroscopy experiments have identified the oxidation states of the Mn in the OEC in each of the intermediate S-states, while extended X-ray absorption fine structure experiments have shown that 2.7 A Mn-Mn di-mu-oxo and 3.3 A Mn-Mn mono-mu-oxo motifs are present in the OEC. X-ray spectroscopy has also been used to probe the two essential cofactors in the OEC, Ca2+ and Cl-, and has shown that Ca2+ is an integral component of the OEC and is proximal to Mn. In addition, dichroism studies on oriented PS II membranes have provided angular information about the Mn-Mn and Mn-Ca vectors. Based on these X-ray spectroscopy data, refined models for the structure of the OEC and a mechanism for oxygen evolution by the OEC are presented.

Sulfur K-edge x-ray absorption spectroscopy: a spectroscopic tool to examine the redox state of S-containing metabolites in vivo.

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii.

Light-activated hydrogen and oxygen evolution as a function of CO2 concentration in helium were measured for the unicellular green alga Chlamydomonas reinhardtii. The concentrations were 58, 30, 0.8 and 0 ppm CO2. The objective of these experiments was to study the differential affinity of CO2/HCO 3 (-) for their respective Photosystem II and Calvin cycle binding sites vis-à-vis photoevolution of molecular oxygen and the competitive pathways of hydrogen photoevolution and CO2 photoassimilation. The maximum rate of hydrogen evolution occurred at 0.8 ppm CO2, whereas the maximum rate of oxygen evolution occurred at 58 ppm CO2. The key result of this work is that the rate of photosynthetic hydrogen evolution can be increased by, at least partially, satisfying the Photosystem II CO2/HCO 3 (-) binding site requirement without fully activating the Calvin-Benson CO2 reduction pathway. Data are presented which plot the rates of hydrogen and oxygen evolution as functions of atmospheric CO2 concentration in helium and light intensity. The stoichiometric ratio of hydrogen to oxygen changed from 0.1 at 58 ppm to approximately 2.5 at 0.8 ppm. A discussion of partitioning of photosynthetic reductant between the hydrogen/hydrogenase and Calvin-Benson cycle pathways is presented.