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A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.

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 x 0.2 x 0.3 mm) of sperm whale MbCO (space group P2(1)) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle (alpha = 23 degrees) or perpendicular (alpha = 86 degrees) 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 similar to 2 eV, due to electronic relaxation of empty states of p(z) 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.