Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and β subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100–200 μM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O₂ buffer and delivery. In contrast, the very low levels of Ngb (~1 μM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O₂ metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

Aims. Nitrobindins (Nbs) are evolutionary conserved all-β-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom. The physiological role(s) of Nbs is almost unknown. Here, the structural and functional properties of the Mycobacterium tuberculosis Nb (Mt-Nb(III)) and Homo sapiens Nb (Hs-Nb(III)) have been investigated and compared with those of Arabidopsis thaliana Nb (At-Nb(III), of the hematophagous bug Rodius prolixus nitrophorins (Rp-NP(III)s), and of mammalian myoglobins. Results. Data here reported demonstrate that Mt-Nb(III), At-Nb(III), and Hs-Nb(III) share with Rp-NP(III)s the capability to bind selectively NO, but display a very low reactivity, if any, towards histamine. Data obtained overexpressing Hs-Nb in HEK293 cells indicate that Hs-Nb localizes mainly in the cytoplasm and partially in the nucleus thanks to a nuclear localization sequence encompassing residues Glu124-Leu154. Human Hs-Nb corresponds to the C-terminal domain of the human nuclear protein THAP4, and our data suggest that Nb may act as a sensor possibly modulating the THAP4 transcriptional activity residing in the N-terminal region. Finally, we provide strong evidence that both Mt-Nb(III) and Hs-Nb(III) are able to scavenge peroxynitrite and to protect dose-dependently free L-tyrosine against peroxynitrite-mediated nitration. Innovation. Data here reported suggest an evolutionarily conserved function of Nbs related to their role as gas sensors and components of the anti-oxidant system. Conclusion. Human THAP4 may act as a sensing protein that couples the heme-based Nb(III) reactivity with gene transcription. Besides, Mt-Nb(III) seems to be part of the pool of proteins required to scavenge RNS and ROS produced by the host during the immunity response.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H₂O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Aims: Structural and functional characterization of the globin-coupled sensors (GCSs) from Azotobacter vinelandii (AvGReg) and Bordetella pertussis (BpeGReg). Results: Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in AvGReg and BpeGReg of a globin domain capable of reversible gaseous ligand binding. In AvGReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and k′_CO is higher than for other GCSs. The O₂ binding kinetics suggests the presence of an open and a closed conformation. As for BpeGReg, the fully oxygenated AvGReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to BpeGReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins (AvGReg globin domain and BpeGRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [BpeGReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, BpeGReg-Gb* shows also a second dimerization mode. Innovation: This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. Conclusions:AvGReg and BpeGReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, AvGReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. BpeGReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.

In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale and can provide precise visual details over a wide size range, from the molecular level up to hundreds of micrometers. The nonperturbative nature, the ability to scan in a liquid environment, and the lack of need for freezing, fixing, or staining make AFM a well-suited tool for studying fragile samples such as macromolecular crystals. Starting from the first morphological investigations revealing the surface morphology of protein crystals, this review discusses the achievements of AFM in understanding the crystal growth processes, both at the micro- and nanoscale. The capability of AFM to investigate the sample structure at the single molecular level is analyzed considering in-depth the structure of S-layers. Lastly, high-speed atomic force microscopy (HS-AFM) is discussed as the evolution to overcome the limitations of low imaging speed, allowing for the observation of molecular dynamics and weakly adsorbed, diffusing molecules. HS-AFM has provided intuitive views and directly visualized phenomena that were previously described indirectly, answering questions that were challenging to address using other characterization methods.

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O₂ dissociation and ultra-high O₂ affinity. However, CerHb affinity for O₂ is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O₂ ligand, the overall binding schemes for CO and O₂ are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.