Stefano Della Longa

Pentacoordinate Hemin Derivatives in Sodium Dodecyl Sulfate Micelles: Model Systems for the Assignment of the Fifth Ligand in Ferric Heme Proteins

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ±(0.02–0.07)-Å accuracy on the atomic distances and ±7° on the Fe–CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.

An X-Ray Diffraction and X-Ray Absorption Spectroscopy Joint Study of Neuroglobin.

Neuroglobin (Ngb) is a member of the globin family expressed in the vertebrate brain, involved in neuroprotection. A combined approach of X-ray diffraction (XRD) on single crystal and X-ray absorption spectroscopy (XAS) in solution, allows to determine the oxidation state and the structure of the Fe-heme both in the bis-histidine and the CO-bound (NgbCO) states. The overall data demonstrate that under X-ray the iron is photoreduced fairly rapidly, and that the previously reported X-ray structure of ferric Ngb [B. Vallone, K. Nienhaus, M. Brunori, G.U. Nienhaus, Proteins 56 (2004) 85-92] very likely refers to a photoreduced species indistinguishable from the dithionite reduced protein. Results from the XAS analysis of NgbCO in solution are in good agreement with XRD data on the crystal. However prolonged X-ray exposure at 15K determines CO release. This preliminary result paves the way to experiments aimed at the characterization of pentacoordinate ferrous Ngb, the only species competent in binding external ligands such as O2, CO or NO.

One-electron excitations and shake up satellites in Cu K-edge X-ray absorption near edge structure (XANES) of La2CuO4 by full multiple scattering analysis in real space

Abstract 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.

Dynamic Investigation of Protein Metal Active Sites: Interplay of XANES and Molecular Dynamics Simulations

The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.

Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to −3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).

Unusual Heme Binding Properties of the Dissimilative Nitrate Respiration Regulator, a Bacterial Nitric Oxide Sensor

AIMS In the opportunistic pathogen Pseudomonas aeruginosa, nitric oxide (NO) triggers the respiration of nitrate (denitrification), thus allowing survival in chronic infection sites as a microaerobic-anaerobic biofilm. The NO-dependent induction of denitrification is mediated by the dissimilative nitrate respiration regulator (DNR), a transcription factor forming a stable complex with heme, which is required to sense the physiological messenger (i.e., NO). The molecular details of NO sensing in DNR and, more in general, in this class of sensors are largely unknown, and a study aimed at integrating microbiology and biochemistry is needed. RESULTS Here we present a comprehensive study, including in vivo results and spectroscopy, kinetics, and protein engineering, that demonstrates the direct involvement of a histidine residue in heme iron coordination. Moreover, a peculiar phenomenon of ligand switching around heme iron, which hampers the identification of the second heme axial ligand, is also suggested. These results indicate that DNR is characterized by a remarkable flexibility in solution, as observed for other cAMP receptor protein/fumarate and nitrate reductase regulators (CRP-FNR) to which DNR belongs. INNOVATION The present work represents one of the few studies focused on the biochemistry of NO sensing by bacterial transcriptional regulators. The data presented demonstrate that structural plasticity of DNR is crucial for the sensing activity and confers to the protein unusual heme binding properties. CONCLUSIONS Protein flexibility and dynamics is a key structural feature essential to explain the evolutionary success and adaptability of CRP-FNR, and may represent a common strategy employed by heme-based redox sensors, which presents features deeply different from those of canonical hemeproteins.

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

X-RAY ABSORPTION SPECTROSCOPY OF HEMOGLOBIN

Publisher Summary This chapter discusses the results obtained by the X-Ray Absorption Spectroscopy (XAS) experimental method on the iron (Fe) site structure of hemoglobin (Hb) and the basic principles of the X-ray absorption spectroscopic method. X-Ray absorption spectroscopy of Hb has provided an experimental method for probing the Fe site structural configurations in solution. The spectral features in the XAS spectra are determined by the scattering of the photoelectrons, emitted by the absorbing Fe ion of Hb and returned to the Fe site by the neighboring atoms within a sphere of about 5 A°. Therefore, the XAS method is a direct probe of atomic distribution via electron diffraction. It probes the spatial distribution of the atoms of the heme, the proximal histidine, and the ligand. The absorption spectrum beyond 5 electron volt (eV) from the edge is because of transitions to high-energy states in the continuum which can be described as due to the scattering of the high-energy photoelectrons excited in the continuum from the neighboring atoms.

Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin

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.