Nitrogen chemical structure in DNA and related molecules by X-ray absorption spectroscopy.

The electronic environment of nitrogen in nucleic acid bases, nucleotides, polynucleotides and DNA has been studied, for the first time using X-Ray Absorption Near-Edge Spectroscopy (XANES). Generally, the spectra of these complex molecules consist of low energy bands corresponding to 1s-->pi* transitions and high energy bands corresponding to 1s-->sigma* transition, as illustrated using several nitrogen model compounds. The 1s-->pi* transitions show particular sensitivity to the chemical environment of the nitrogen. Oxygen substitution on ring carbon atoms generally results in a significant blue shift of the lowest 1s-->pi* bands while halogen substitution results in a small blue shift. These observations illustrate the significance of the disturbance of the aromatic ring system produced by exocyclic carbonyl groups. Direct substitution on the nitrogen frequently results in significant spectral perturbations. Differences between the spectra of the polynucleotides and the sums of spectra of the individual nucleotides point to the effects of hydrogen-bonding in complementary double-helix structures. The XANES spectrum of a DNA sample with a known ratio of the polynucleotides is equivalent to the weighted sum of the spectra of individual polynucleotides, indicating that the difference in base stacking interactions produces negligible spectral effects. The variability of nitrogen K-edge spectra in these samples and in protein may be useful for chemically specific imaging using X-ray microscopes.     

Structure of the Fe-heme in the homodimeric hemoglobin from Scapharca inaequivalvis and in the T72I mutant: an X-ray absorption spectroscopic study at low temperature

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.     

Photoabsorption study of Bacillus megaterium, DNA and related biological materials in the phosphorus K-shell edge region

We measured the X-ray transmission spectra of several biologically related samples in the phosphorus K-shell edge absorption region. These include red phosphorus, hydrated sodium phosphate (Na(3)PO(4).12 H(2)O), deoxyribonucleic acid (DNA), adenosine triphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position: Each is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-shell edge absorption thresholds are shifted toward higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B. megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA, ribonucleic acid (RNA) and phosphates within the spore. The B. megaterium spore spectrum provides information for resonant radiation damage studies in the phosphorus K-shell edge absorption region by identifying candidate photoexcitations. In addition, the absorption spectra will be useful in X-ray microscopy and macromolecular crystallography studies at the phosphorus K-shell edge.     

Structural and electronic characterization of heme moiety in oxygenated hemoproteins by using XANES spectroscopy.

Iron K-edge X-ray absorption near edge structure (XANES) spectra were measured for oxy-forms of cytochrome P-450cam (P-450cam), horseradish peroxidase (HRP) and myoglobin (Mb) by using Synchrotoron Radiation of Photon Factory (Tsukuba). A pronounced 1s-4p transition and some fine structures were well-resolved in the spectra obtained. Comparing the spectra, the features at the fine structures termed P, C and D, were similar among the three hemoproteins, suggesting a similar site-symmetry around the heme iron and the same Fe-O-O bond angle (about 115 degrees). On the other hand, absorption features at the edge region (7115-7135 eV) were slightly but significantly different from one another; the absorption intensity at 7115-7125 eV region increased in the order of Mb, HRP and P-450cam, while that at 7125-7135 eV decreased in the same order. A similar absorption feature was also obtained with their deoxy (ferrous high spin) forms. We assumed that the absorption at the lower energy region (7115-7125 eV) reflects the pi-character in the Fe-ligand bond, whereas that at the higher energy region (7125-7135 eV) does the sigma-character, on the basis of the previous and comprehensive studies of the XANES spectroscopy of the adsorbed molecules on the metal surface (McGovern et al. (1989) Handbook on Synchrotoron Radiation, Vol. 2, pp. 467-539). According to our assumption, our XANES results indicated that the pi-character of the Fe-ligand bond increases in the order of Mb, HRP and P-450cam, and that the pi-electron of the thiolate S- in P-450cam is donated to the Fe-O-O moiety, most probably to the antibonding pi* orbital of O2. Such an interpretation is consistent with the experimental findings or data accumulated so far by other methods, such as the resonance Raman spectroscopy.     

Ion desorption from DNA components irradiated with 0.5 keV ultrasoft X-ray photons

Positive ion desorption from thin films of DNA components, 2-deoxy-d-ribose, thymine, thymidine (dThd), and thymidine 5'-monophosphate (dTMP) was investigated in the oxygen K- shell edge excitation region using synchrotron ultrasoft X rays (538 eV). A large number of molecular fragments, H(+), CH(x)(+), C(2)H(x)(+), CO(+), CH(x)O(+), C(3)H(x)(+), C(2)H(x)O(+) and C(3)H(x)O(+) (x = 1, 2 and 3), were observed as desorbed ions from 2-deoxy-d-ribose. Some of these ions are related to simultaneous bond scission at particular C-C and C-O (or C-C) bonds in the furanose ring structure in the 2-deoxy-d-ribose molecule, indicating that the impact of photons on the oxygen atom and the impact of ejected secondary electrons (e.g. Auger electrons) cause an intense destruction of the furanose ring structure. In thymine thin films, H(+), CH(x)(+), CO(+), CH(x)O(+), C(2)H(x)N(+) and CH(x)NO(+) (x = 1, 2 and 3) fragments were observed. The yields of these ions were smaller than the yields from 2-deoxy-d-ribose. The desorption of CH(3)(+) from thymine might induce a molecular conversion from thymine to uracil. The mass patterns of dThd and dTMP, and especially that of dTMP, were similar to that of 2-deoxy-d-ribose, indicating that a number of ions were generated at the sugar site, even in the nucleotide molecule. It is therefore predicted that the sugar moiety is more fragile than the thymine base.     

Spin-resolved X-ray absorption near edge structure (XANES) simulation of metmyoglobin

Spin resolved multiple scattering (MS) calculations of Fe K-edge X-ray Absorption Near Edge Structure (XANES) of myoglobin are reported for the first time. The observed differences of the Fe K-edge XANES spectra of sperm whale metmyoglobin under spin transition as a function of temperature have been studied. The method allows one to compute separately spin effects and local structural effects. The results show that spin effects are confined in the absorption rising edge in the range 7111–7130 eV, while purely structural effects are dominant in the range 7130–7170 eV. Symmetry changes of the Fe coordination sphere mainly related to its movement towards the heme plane, coupled to an increase of axial asymmetry, can explain the XANES changes observed above 7130 eV without an appreciable change of the Fe-N_p distance. Received: 8 October 1997 / Revised version: 10 March 1998 / Accepted: 23 March 1998     

XANES study of iron displacement in the haem of myoglobin

The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry.     

Oxygen bonding in human hemoglobin and its isolated subunits: a XANES study

The X-ray absorption near edge structure (XANES) spectra of the human adult and foetal hemoglobin, of the isolated alpha and beta chains, in the oxygenated forms, and of the oxymyoglobin and carp oxyhemoglobin have been measured at the wiggler beam line of the Frascati Synchrotron radiation facility. The bonding angle of oxygen molecule at the iron site in these hemoproteins in solution, has been measured using the multiple scattering theory for data analysis.     

Damage to hemoglobin by radiation-generated serum albumin radicals.

We have studied the effects of the interaction of radiation generated human serum albumin radicals (HSA*) with human hemoglobin molecules (Hb). Diluted Hb aqueous solutions were irradiated under N2O or argon without HSA and in the presence of HSA. Analysis of Hb absorbance spectra in the visible range, cross-linking of HSA* radicals with Hb molecules and functional properties of Hb were investigated. The degree of Hb destruction estimated on the basis of changes in the absorption spectra indicated that the effectiveness of HSA* radicals generated under N2O for Hb destruction was approximately equal to that of *OH radicals. In this case mainly *OH radicals formed the secondary HSA* radicals. However, during the irradiation Hb + HSA under argon the presence of equivalent amounts of oxidizing and reducing products of water radiolysis lowers the degree of Hb destruction. Some reactions of HSA* radicals with Hb molecules lead to the formation of covalent bonds between the molecules of both proteins. The following types of hybrids could be distinguished: Hb monomer-HSA, Hb dimer-HSA and higher aggregates. Structural changes of Hb by HSA* radicals were reflected by alterations in the oxygen affinity (increase) and cooperativity (decrease) of Hb. The results obtained indicate that in the experimental systems studied, the HSA* radical reactions with Hb molecules are favoured over recombination reactions of HSA* radicals. On this basis one can suggest that in the studied systems Hb plays the role of an acceptor of radical energy located on HSA.     

Evaluation of the energetic position of the lowest excited singlet state of beta-carotene by NEXAFS and photoemission spectroscopy.

In carotenoids the lowest energetic optical transition belonging to the pi-electron system is forbidden by symmetry, therefore the energetic position of the S(1) (2(1)A(g)) level can hardly be assessed by optical spectroscopy. We introduce a novel experimental approach: For molecules with pi-electron systems the transition C1s-->2p(pi*) from inner-atomic to the lowest unoccupied molecular orbital (LUMO) appears in X-ray absorption near edge spectra (NEXAFS) as an intense, sharp peak a few eV below the carbon K-edge. Whereas the peak position reflects the energy of the first excited singlet state in relation to the ionization potential of the molecule, intensity and width of the transition depend on hybridization and bonding partners of the selected atom. Complementary information can be obtained from ultraviolet photoelectron spectroscopy (UPS): At the low binding energy site of the spectrum a peak related to the highest occupied molecular orbital (HOMO) appears. We have measured NEXAFS and UPS of beta-carotene. Based on these measurements and quantum chemical calculations the HOMO and LUMO energies can be derived.     

Mn K-edge XANES spectroscopy of a photosynthetic O2-evolving complex. High-quality pre-edge features and distinct fine structures in the S1- and S2-states

High-resolution XANES (X-ray Absorption Near Edge Structure) spectroscopy for Mn in the S1 and S2 states of the spinach photosynthetic O2-evolving complex revealed distinct features in K-edge spectra, when a high signal-to-noise (S/N) ratio of ca. 80 with a low and constant background-to-signal (B/S) ratio of 0.15 to 0.18 was attained. Six features resolved in each S-state spectrum involve a pre-edge feature due to 1s----3d transitions, a main-edge feature possibly due to 1s----4s transitions and four fine structures superimposed on the principal absorption bands due to 1s----4p* transitions. The high-quality pre-edge features were analyzed according to a parametric ligand-field theory in comparison with those of some typical authentic Mn complexes. It was deduced that i) all of the four Mn ions in the S1-state are octahedrally coordinated and two of them constitute a di-mu-oxo bridged Mn(III, III) dimeric subunit; ii) the bridged Mn(III) ions are further bridged by a deprotonated water dimer, (HOHOH)-, and coordinated by imidazole-N and carboxylate-O- on the opposite side of the Mn atom from the di-mu-oxo bridge; iii) the other two Mn ions exist in the form of Mn(III) monomeric subunits; and iv) upon the S1----S2 transition, only the bridged Mn(III,III) is oxidized to Mn(III,IV). The distinct change in the principal absorption band shape upon the S1----S2 transition is briefly discussed to obtain the XANES evidence for a tetrameric Mn-cluster.     

Chloride ligation in inorganic manganese model compounds relevant to Photosystem II studied using X-ray absorption spectroscopy

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures Mn^IV₃O₄X (X=Cl, F, or OH) that contain a di--oxo and two mono--oxo bridges or Mn^IV₂O₂X (X=Cl, F, OH, OAc) that contain a di--oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X=O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3 Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0520-1Abbreviations bpea N,N-bis(2-pyridylmethyl)ethylamine - EXAFS extended X-ray absorption fine structure - FT Fourier transform - IPE inflection point energy - OEC oxygen evolving complex - PS II Photosystem II - tacn 1,4,7-triazacyclononane - XANES X-ray absorption near edge structure - XAS X-ray absorption spectroscopy - XRD X-ray diffraction     

The Five-To-Six-Coordination Transition of Ferric Human Serum Heme-Albumin Is Allosterically-Modulated by Ibuprofen and Warfarin: A Combined XAS and MD Study

Human serum albumin (HSA) is involved physiologically in heme scavenging; in turn, heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, the allosteric effect of ibuprofen and warfarin on the local atomic structure around the ferric heme-Fe (heme-Fe(III)) atom of HSA-heme-Fe (HSA-heme-Fe(III)) has been probed by Fe-K edge X-ray absorption spectroscopy (XAS). The quantitative analysis of the Fe-K edge extended X-ray absorption fine structure (EXAFS) signals and modeling of the near edge (XANES) spectral features demonstrated that warfarin and ibuprofen binding modify the local structure of the heme-Fe(III). Combined XAS data analysis and targeted molecular dynamics (MD) simulations provided atomic resolution insights of protein structural rearrangements required to accommodate the heme-Fe(III) upon ibuprofen and warfarin binding. In the absence of drugs, the heme-Fe(III) atom is penta-coordinated having distorted 4+1 configuration made by the nitrogen atoms of the porphyrin ring and the oxygen phenoxy atom of the Tyr161 residue. MD simulations show that ibuprofen and warfarin association to the secondary fatty acid (FA) binding site 2 (FA2) induces a reorientation of domain I of HSA-heme-Fe(III), this leads to the redirection of the His146 residue providing an additional bond to the heme-Fe(III) atom, providing the 5+1 configuration. The comparison of Fe-K edge XANES spectra calculated using MD structures with those obtained experimentally confirms the reliability of the proposed structural model. As a whole, combining XAS and MD simulations it has been possible to provide a reliable model of the heme-Fe(III) atom coordination state and to understand the complex allosteric transition occurring in HSA-heme-Fe(III) upon ibuprofen and warfarin binding.     

X-ray absorption near edge structure (XANES) for CO, CN and deoxyhaemoglobin: geometrical information

We use the recently developed multiple scattering theory to give a quantitative analysis of the X-ray absorption near edge structure (XANES) of haemoglobin and some of its substituents. We demonstrate that the XANES may contain information not provided by the extended X-ray absorption fine structure (EXAFS) part of the spectrum about the coordination geometry around the Fe atom, and in particular discuss the sensitivity of the XANES to the orientation of the CN group in HbCN. The anisotropy of the system leads to a strong dependence of the calculated spectrum on the polarisation of the X-rays. We show how this effect can be exploited in further XANES structural studies.     

Free radical production by hydroxy-salen manganese complexes studied by ESR and XANES

Three salen-Mn(II) complexes bearing hydroxyl groups in either the ortho, para or meta positions have been synthesized and the structures of the metal complexes and their potential to produce free radicals investigated by electron spin resonance (ESR) and X-ray absorption near edge structures (XANES) spectroscopy. All three compounds were shown to generate a high level of superoxide anions in dimethyl sulfoxide (DMSO) solution. The production of oxygen radicals results from a one electron process oxidation of Mn(II) species leading to the formation Mn(III) redox state species, as revealed by a higher XANES edge energy of 2.7 eV. The formation of superoxide anion was characterized by ESR, both directly and via the use of a spin-trapping method. Under reductive condition in the presence of ascorbic acid, the reduction of Mn(III) to Mn(II) leads to the production of hydroxyl radicals by the ortho and para compounds. The efficient production O(2)*- by such salen-Mn complexes could be useful to evaluate the scavenging properties of antioxidant molecules.     

Investigation of Elemental Sulfur Speciation Transformation Mediated by Acidithiobacillus ferrooxidans

The speciation transformation of elemental sulfur mediated by the leaching bacterium Acidithiobacillus ferrooxidans was investigated using an integrated approach including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray absorption near edge spectroscopy (XANES). Our results showed that when grown on elemental sulfur powder, At. ferrooxidans ATCC23270 cells were first attached to sulfur particles and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to polysulfides. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested that these globules consisted of elemental sulfur bound to thiol groups of protein. Huan He and Cheng-Gui Zhang made equal contributions to this paper.     

Mapping and Measuring DNA to Protein Ratios in Mammalian Sperm Head by XANES Imaging

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.     

Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy

X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.     

Changes in Fe site structure from fetal to adult hemoglobin probed by XANES

Iron X-ray absorption near edge structure (XANES) spectra of human fetal (F) and adult (A) deoxyhemoglobin (deoxyHb) measured at the Frascati synchrotron radiation facility reveal the different geometrical structure of the Fe-porphyrin complexes in the two proteins. By this method, having determined for the first time the variation of atomic positions in fetal and adult hemoglobin in solution (close to the 'in vivo' situation), we give further insight into the structure-function relationship in hemoglobins.     

In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscopy.

During the oxidation of sulfide and thiosulfate purple and green sulfur bacteria accumulate globules of 'elemental' sulfur. Although essential for a thorough understanding of sulfur metabolism in these organisms, the exact chemical nature of the stored sulfur is still unclear. We applied sulfur K-edge X-ray absorption near edge spectroscopy (XANES) to probe the forms of sulfur in intact cells. Comparing XANES spectra of Allochromatium vinosum, Thiocapsa roseopersicina, Marichromatium purpuratum, Halorhodospira halophila and Chlorobium vibrioforme grown photolithoautotrophically on sulfide with reference probes (fingerprint method), we found sulfur chains with the structure R-S(n)-R. Evidence for the presence of sulfur rings, polythionates and anionic polysulfides in the sulfur globules of these bacteria was not obtained.