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The Fe(III) → Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T>160K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02–0.06-Å accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15Å, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-Å statistical error.

The Fe site structure in the recombinant wild-type and T72I mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca inaequivalvis 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.

The Cu site structure of human serotransferrin and hen ovotransferrin using XANES spectroscopy has been investigated. Although the transferrin family proteins have been extensively studied, the results reported herein are the first concerning the structure of the metal site in C-terminal and N-terminal in the whole protein. Our structural data show that these proteins differ with regard to the independence of the two binding sites and the geometry of copper coordination, ranging from a poorly to a significantly distorted octahedron.

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.

The linearly polarized Cu L3-edge X-ray absorption near edge structure (XANES) of a La2CuO4 single crystal has been measured and the spectrum is interpreted by the full multiple scattering approach in the real space. The polarized XANES spectra over a range of 20 eV can be predicted in term of a one-electron dipole (δl = +1) transitions Cu 2p→ϵd, probing the unoccupied d-like (l=2) density of states of the high energy conduction bands. The white line is shown to be due to Cu 2p→3d transitions giving a bound final state Cu 2p53d10 formed by the 5.5 eV Coulomb interaction between the core hole and the 3d electron. The probability of ml=2 orbital angular momentum for the localized Cu 3d hole is shown to be in the range 95–100%.

The linearly polarized Cu {ital L}{sub 3}-edge x-ray-absorption near-edge structure (XANES) of Bi{sub 2}Sr{sub 2}CaCu{sub 2}O{sub 8+{delta}} has been measured and the spectra are interpreted by the full multiple-scattering approach in real space. The polarized spectra over a range of 20 eV can be predicted in terms of the one-electron dipole ({Delta}{ital l}=+1) transition Cu 2{ital p}{r arrow}{epsilon}{ital d}, probing the unoccupied {ital d}-like ({ital l}=2) density of states projected on the Cu site with orbital angular momentum {ital m}{sub {ital l}}=0, 1 in the {bold E}( ){ital z} spectra, and the {ital m}{sub {ital l}}=2, 1, and 0 in the {bold E}{perpendicular}{ital c} spectra. The oscillator strength for the dipole allowed transitions ({Delta}{ital l}={minus}1) Cu 2{ital p}{r arrow}{var epsilon}{ital s} is shown to be a factor of 100 weaker than the 2{ital p}{r arrow}3{ital d} transitions. The Coulomb interaction in the final state between the Cu 2{ital p} core hole and the excited Cu 3{ital d} electron is found to be 5.5 eV forming a bound state below the continuum threshold, the well-known Cu {ital L}{sub 3} white line. On the contrary, the core hole induces a nearly rigid redshift about 1 eV of the high-energy conduction bands.

The polarized Cu K-edge X-ray Absorption Near Edge Structure (XANES) of La2CuO4 has been interpreted by the multiple scattering approach. The size of the cluster of neighbouring atoms having good agreement with the XANES experimental data is determined by 45 atoms surrounding the absorbing Cu. The polarized spectra can be predicted in term of a one-electron dipole (Δl=+1) transition Cu 1s→ϵp, probing the unoccupied p-like (l=1) density of states projected on a Cu site with orbital angular momentum ml=0 in the E∥z spectra, and the ml=1 for the E⊤c spectra. Thus we show that the electronic structure of the high energy conduction bands, beyond the Cu 3d band, of La2CuO4 are well described in terms of the one-electron approximation. It is shown that XANES spectra are consistent with the contraction of the Cu-apex oxygen distance with doping. Final state effects induced by the core hole have been indentified: (i) the core transitions take place in the fully relaxed potential, (ii) the satellite at 7 eV above the main K-XANES peak in both polarizations is assigned to a multielectron shake up excitation. Finally the shoulder on the rising absorption edge, present in many Cu compounds and usually assigned to a shake down multi-electron excitation, is shown to be due to a one-electron transition to a state delocalized over a large cluster.

XANES spectra of non structural protein 3 (NS3) have been calculated using 4 Zn coordination models from three crystallographic structures in the Protein Data Base (PDB): 1DY9, subunit B, 1CU1 subunit A and B, and 1JXP subunit B. Results indicate that XANES is an appropriate tool to distinguish among them. Experimental XANES spectra have been simulated refining crystallographic data. The model obtained by XAS is compared with the PDB models.

We investigated for the first time the haem stereochemistry in the nitrosylated derivative of two amphibian haemoglobins, Xenopus laevis and Ambystoma mexicanum, by means of X-ray absorption spectroscopy technique with the aim to explain the relationships between the active site structure and physiological function of these proteins, compared to that from humans. Our results show that while the Fe site local structure of human HbNO is modulated by an allosteric effector such as IHP shifting the T-R equilibrium towards the T-state, the Fe site local structure of amphibians HbNO is stabilized in a particularly tensed T-state also without IHP.

The changes of the Fe heme-active site conformation of dromedary (Camelus dromedarius) nitrosylhemoglobin (HbNO) induced by inositol hexakisphosphate (IHP) and chlofibric acid (CFA) have been studied by using X-ray absorption near-edge structure (XANES) spectroscopy. Structural information has been determined by multiple scattering analysis of the Fe K-edge XANES spectra. The proximal histidine is found to move away from iron centers by about 0.4 Angstrom on the average over the four hemes upon binding of CFA or stoichiometric amount of IHP. In molar excess of polyanion or in the simultaneous presence of IHP, CFA and chloride, the proximal histidine moves back to a position very close to that observed in pure buffer; yet, the structure modulation induced by the allosteric effectors is not completely reversible. Such findings parallel with the functional properties and the spectroscopic (e.g., EPR and absorbance) characteristics of HbNO.

The active metal site structure of transferrin with iron and copper atoms is investigated using metal K-XANES. Theoretical analysis of experimental data has been performed on the basis of full multiple-scattering theory. This approach made it possible to study the origin of XANES fine details and to investigate the local structure around active metal sites. A deep insight into the local structure and electronic subsystem of Fe, Cu transferrins is obtained. For example, in the case of Cu substitution of Fe in the active centre, the best fit of theoretical spectra to experiment has been obtained for distances 3% smaller between the Cu atom and the nearest neighbours.

A new software procedure, MXAN, to fit experimental XANES spectra is presented here. The method is based on the comparison between the experimental spectrum and several theoretical calculations generated by changing the relevant geometrical parameter of the site around the absorbing atom. The x-ray photoabsorption cross section is calculated using the general multiple-scattering scheme, utilizing a complex Hedin-Lunqvist energy-dependent potential to describe the exchange correlation interaction. Our method has been applied to the study of geometrical environment of the tetrahedral zinc site of the protein superoxide dismutase (SOD). The experimental Zn K-edge XANES spectrum has been fitted in the space of the first shell coordination parameters (ligand distances and angles) following the behavior of the chi-square as a function of the local distortion from the starting crystallographic structure. The recovered structure is found to be independent on the starting conditions, showing the theoretical uniqueness of the structural solution. Strengths and limitations of the application to real systems are also discussed.

Visible light can break the Fe—CO bond in Fe(II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T < 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T = 20 K) of an oriented single crystal (0.2 × 0.2 × 0.3 mm) of sperm whale MbCO (space group P21) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle α = 23°) or perpendicular (α = 86°) to the heme normal of each myoglobin molecule. The crystal was continuously illuminated by a white-light source during the data collection. The polarized data give novel information on the Fe-heme electronic/structural rearrangement following photolysis. The XANES (X-ray absorption near-edge structure) spectrum polarized in the direction close to the Fe—CO bond changes dramatically after photolysis, exhibiting a shift of ∼2 eV, due to electronic relaxation of empty states of pz symmetry, while more subtle changes are observed in the spectrum polarized along the heme plane, sensitive to the heme-plane geometry. Changes in the pre-edge region can be interpreted to provide insight into the electronic structure of the highest occupied and lowest unoccupied molecular orbitals (HOMO–LUMO) in the MbCO → Mb* photochemical reaction at low temperature.

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-Np distance.

We have measured the x-ray absorption near edge structure (XANES) spectra of the enzyme tyrosinase from the mushroom Agaricus bisporus in solution in the oxy and deoxy forms. The spectra, obtained under the same conditions as the analogous forms of mollusc hemocyanin (Hc), show that the oxidation state of copper changes from Cu(II) (oxy form) to Cu(I) (deoxy form), and the copper active site(s) of A. bisporus tyrosinase in solution undergoes the same main conformational changes as Hc. We have applied the multiple scattering theory to simulate the XANES spectra of various alternative geometries of the copper site, accounting for the residual differences between Hc and tyrosinase. While oxy-Hc is reasonably fitted only by the pseudo-square-pyramidal geometry reported by its crystallographic data, oxytyrosinase can be fitted, starting from the Hc coordinates, either by distortions toward a pseudo-tetrahedral geometry, with inequivalent copper sites, or by an apically distorted square-pyramidal geometry (with an elongation of the apical distance of no more than 0.2 A).

The ligand photodissociation of sperm whale carboxymyoglobin (MbCO) at low temperature (15K-100K) under extended illumination has been studied by X-ray Absorption Near Edge Structure (XANES) spectroscopy using the dispersive technique. XANES simulations through the multiple scattering (MS) approach allow one to interpret the spectroscopic data in structural terms, and to investigate the Fe site structure configurations of the states that follow the CO photodissociation as a function of temperature. The Fe site in the photoproduct is unbound, with an overall structure similar to the deoxy-form (Mb) of the protein. The Fe site structure changes from T30K(Mb*) to T50K (Mb**), revealing the existence of a slower unbound state Mb**. A model is proposed which includes the faster state (Mb*) as a planar porphyrin ring with a displacement of Fe from the heme plane of less than 0.3 A, and the slower state (Mb**) with a domed heme.

The structural changes of the Fe active site following the low temperature photodissociation of sperm whale carboxymyoglobin (MbCO) has been studied by X‐ray Absorption Near Edge Structure (XANES) with the dispersive X‐ray absorption method. This experimental method allows to study time dependent biological processes in diluted samples in the time scale of 10–100 seconds. From the Fe K‐edge XANES difference spectra between the MbCO and its photoproduct (Mb*), a model of the intermediate state Mb* at low temperature (15 K) can be inferred. The structure of the photoproduct Mb* is non‐ligated, with the iron out of the heme plane, but distinct from the deoxy state Mb. A structural model built taking into account EXAFS and Raman data on Mb* explains some but not all the features observed by XANES spectroscopy. A smaller displacement of the iron from the pyrrolic nitrogen plane, coupled to a more domed structure of the porphyrin plane could explain better the spectral differences between the Mb* and Mb states.

Geometry structure optimization of the potential antitumor agent Au(bipy)(OH)2 was carried out by means of density functional theory simulations. The experimental Au L3-edge X-ray absorption near edge structure (XANES) spectrum of Au(bipy)(OH)2 was obtained. The theoretical Au L3-XANES spectra of the gold(III) complex Au(bipy)(OH)2 were simulated using both the self-consistent real-space full multiple scattering theory within the muffin-tin approximation for the potential shape and the full-potential finite difference method. The comparison of the theoretical spectra with the experimental XANES is discussed. The exact local atomic structure of gold complex Au(bipy)(OH)2 has been defined by two independent ab initio methods.

The active site structure of the oxygenated derivative of the main subassemblies (whole protein, dodecamers, and trimers) of the giant haemoglobin from Eisenia foetida has been characterized by x-ray absorption near edge structure spectroscopy. The data revealed a remarkable effect of the hierarchic assemblies on the active site of the subunit. Specifically, the whole protein has the same site structure of the dodecamer, while a sharp conformational transition occurs when the dodecamer is disassembled into trimers (and monomers) revealing that constraints due to the protein matrix determine the active site geometry and, consequently, the protein function in these large complexes.

The iron site structure modifications induced by the transition from the quaternary R to T structure in ferric carp azide hemoglobin have been detected from analysis of multiple scattering resonances in the XANES (X-ray absorption near edge structure) spectra. High signal-to-noise XANES spectra measured at the Frascati ‘wiggler’ synchrotron radiation facility reveal that the forces on the Fe active site, due to the transition from the R to T quaternary conformation, only induce the tilting of the porphyrin plane and probably also of the proximal histidine. The variation of the Fe-N mean distance is not detected by XANES spectroscopy and therefore it is less than 0.01 A.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

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.

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.

X-ray absorption near edge structure (XANES) spectra of a model compound chelated protoheme methyl ester in solution reveal a linear Fe-C-N configuration and a bent Fe-C-O configuration, although there are no distal steric effects.

Cu L3 x-ray absorption near edge structure (XANES) and Cu L 3 x-ray photoelectron spectroscopy (XPS) of YBa2Cu3O6.5+x are compared. The breakdown of one-electron picture of its electronic structure is reported. The data are interpreted by mixing of Cu 3d9 and of [Formula: see text] (where [Formula: see text] is a hole in the oxygen derived band, ligand hole) many body configuration in the initial state. The localization of Cu 3d9 configuration is indicated by the bare Coulomb interaction Udd~6 eV . The conductivity is assigned to the itinerant [Formula: see text] configuration. The experimental evidence that the additional oxygen x, giving higher Tc , increases the weight of the [Formula: see text] configuration is reported. The presence of holes on the oxygen atoms is confirmed by the Ols XPS spectra. The Cu3+(Cu 3d8) configuration is not observed in L3 XANES in agreement with valence band XPS giving the energy of the 3d8 excited state at about 12 eV above the ground state. An energy scheme of the many body configurations in YBa2Cu3O~7 is obtained. These experiments give experimental evidence that the high Tc superconductivity is due to pairing of holes in the oxygen valence band interacting with localized electrons at the Cu sites.

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.

Our knowledge of the structure of condensed matter has been based primarily on spectroscopic methods that measure first-order pair correlations of atomic arrangements and thus provide interatomic distances (for example neutron and X-ray scattering). Bond angles are given by higher-order correlation functions, and such information can be provided by X-ray absorption near-edge structure (XANES) spectroscopy, the features of which are determined by multiple scattering of photoelectrons whose paths begin and end at the selected absorbing atom. We report here angular-resolved XANES spectroscopy of a single crystal of carboxymyoglobin (MbCO). The large dichroism of the X-ray absorption of the crystal can be fully interpreted by multiple-scattering theory which allows the determination of Fe-ligand bond angles. The analysis of the identified multiple scattering features due to CO in high signal-to-noise-ratio spectra of protein in solution has allowed the determination of the variation of CO bond angles. This opens the way to the determination of subtle structural features due to bond angle variations in proteins in solution which are relevant to an understanding of the characteristics of proteins at the atomic scale.

Cooperativity is a fundamental problem in molecular biology. Camelus Dromedarius hemoglobin is known to assume different functional and structural states as a function of the presence of same polyanionic allosteric effectors. We have studied the local structure of the CO-bound Fe site in this hemoglobin in the presence of inositolexaphosphate (IHP), chlofibric acid (CFA) and Dl-. Our results indicate that while IHP and the simultaneus presence of IHP and CFA has almost no effect on Fe site, the presence of CFA and of all the allosteric effectors induces a distortion on the heme consistent with a lower Fe-CO angle and a reduced Fe-Np distance.

The ligand bonding geometry of carboxy- and cyanomet-myoglobin (MbCO and MbCN) has been measured by the XANES method (X-ray Absorption Near Edge Structure). A comparison between the ligand bonding geometry of carboxy- and cyanomet-myoglobin and of chelated protoheme methyl ester shows that the bent Fe-C-O configuration is the same in both systems. Therefore, we suggest that this configuration is not associated with any steric constraint imposed by the side chains of the aminoacid residues at the distal side of the heme pocket.

The Fe-site structure variation in the transition from the low-affinity tense (T) quaternary structure to the high-affinity relaxed (R) structure in carp deoxyhemoglobin was studied by analysis of multiple scattering resonances in the XANES (x-ray absorption near edge structure) spectra. High signal-to-noise XANES spectra were measured at the Frascati "wiggler" synchrotron radiation facility. We find that the forces on the Fe active site due to the change of quaternary protein conformation do not induce variations greater than 0.01 A in interatomic Fe-N distances, variations greater than 0.1 A in the Fe displacement toward the heme plane, or the "doming" of the heme. The relevance of these results to the mechanism of protein control of ligand binding is discussed.

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.

The bonding angle of oxygen and carbonmonoxy molecules in oxygen carrier hemoproteins is a key parameter to describe the bond strength of the diatomic molecules to iron atom and therefore to understand the mechanism of reversible bonding and releasing of oxygen in hemoglobin. In spite of the large number of studies of haemoglobin the determination of oxygen bonding angle in the proteins in solution escapes the available experimental methods. A different bonding angle of oxygen in oxy-hemoglcbin (HbO2) and in oxy-myoglobin (MbO2) single crystals has been recently reported [1]. The different bonding angle in the two hemoproteins which have different biological roles, transport and storage of oxygen molecules respectively, but similar local structure at the iron site has renewed the interest in this problem. We report the first application of the XANES (X-ray Absorption Near Edge Structure) spectroscopy to obtain information on the oxygen bonding geometry in the protein in solution (close to the “in vivo” situation) which cannot be studied by diffraction methods. The appealing aspect of XANES to study this problem is that the multiple scattering resonances of the photoelectron emitted at the iron site in the 10–50 eV energy range depend on the relative atomic positions of neighbour atoms and not only on the first order radial distribution function like EXAFS (Extended X-ray Absorption Fine Structure), therefore it is a direct structural probe of bonding angles [2–4].

Fast energy dispersive x-ray spectroscopy has been used for in-situ time dependent observations of irreversible structural changes of carboxy-hemoglobin in solution. For the first time one is able to proceed with both in situ and time-dependent investigations of the changes of the local structure by XANES (x-ray absorption near edge structure) of of the active site of a protein during a chemical process.

The copper L2,3 soft x-ray absorption spectra of the single phase high temperature (Tc=91 K) superconductor YBa2Cu3O≈7 have been measured by using synchrotron radiation at LURE. The spectra show at the absorption threshold a white line at 931.1 eV due to Cu 2p 3d10 final states (the underline indicates the hole) for the Cu 3d9 initial state, and a broad high energy shoulder with the maximum at 933 eV assigned to Cu 2p 3d10 L and to Cu 2p 3d10 L 4s1 final states ( L indicates a hole in the oxygen 2p derived valence band), i.e. to the initial states 3d9 L . The intensity of this last feature has been found to decrease with decreasing oxygen content x in YBa2Cu3O6.5+x. No evidence of Cu 2p 3d9 final states has been found. This experiment gives direct evidence that additional oxygen x does not give Cu3+ ions with Cu 3d8 configuration but gives 3d9 L electronic configuration (i.e. Cu2+O- pairs) and no gap is observed between the 3d9 (Cu+) and 3d10 L configuration (i.e. Cu+O- pairs). Therefore in the metallic phase which exhibits superconductivity a continous set of itinerant states 3d9 L and 3d10 L is observed.

The experimental determination of the bonding geometry of the diatomic molecules O2, CO and CN in hemoproteins in solution is still an open problem in spite of extensive experimental research. Diffraction experiments are limited to crystallized proteins, on the contrary x-ray absorption near edge structure (XANES) spectroscopy is a tool for determination of local structure of active site in metalloproteins in solution(1–8).

The Fe-heme structure of hemoglobin under various 'in vitro' denaturing conditions has been investigated by X-ray Absorbption Near Edge Structure (XANES) spectroscopy at the Fe K-edge. The Fe 3+ ion in denatured hemoglobin is found both in low-spin and high-spin rhombic fields. New structural information on the low-spin forms (hemichromes), complementary to that already furnished by Electron Paramagnetic Resonance (EPR), is achieved. The presence and relevance of these denatured forms in the 'in vivo' process of red cell aging is discussed.

The x-ray absorption near edge structure (XANES) spectra of hemoglobin and myoglobin have been measured at the wiggler beam line of the Frascati Synchrotron Radiation Facility. The energy shifts of the iron absorption jump edge and the chemical shifts of the bound excited state at threshold of 1s core excitations, going from deoxygenated to oxygenated form, are interpreted as evidence of some increase of the positive effective charge on the iron atom upon oxygenation.