Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties.

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.

Mycobacterial and Human Nitrobindins: Structure and Function.

Aims: Nitrobindins (Nbs) are evolutionary conserved all-ß-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 ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) and ferric Homo sapiens Nb (Hs-Nb(III)) have been investigated and compared with those of ferric Arabidopsis thaliana Nb (At-Nb(III), Rhodnius prolixus nitrophorins (Rp-NP(III)s), and 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 nitric oxide, but display a very low reactivity, if any, toward histamine. Data obtained overexpressing Hs-Nb in human embryonic kidney 293 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 suggesting 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 free l-tyrosine against peroxynitrite-mediated nitration. Innovation: Data here reported suggest an evolutionarily conserved function of Nbs related to their role as nitric oxide sensors and components of antioxidant systems. Conclusion: Human THAP4 may act as a sensing protein that couples the heme-based Nb(III) reactivity with gene transcription. Mt-Nb(III) seems to be part of the pool of proteins required to scavenge reactive nitrogen and oxygen species produced by the host during the immunity response.

Surface Enhanced Raman Spectroscopy for In-Field Detection of Pesticides: A Test on Dimethoate Residues in Water and on Olive Leaves.

Dimethoate (DMT) is an organophosphate insecticide commonly used to protect fruit trees and in particular olive trees. Since it is highly water-soluble, its use on olive trees is considered quite safe, because it flows away in the residual water during the oil extraction process. However, its use is strictly regulated, specially on organic cultures. The organic production chain certification is not trivial, since DMT rapidly degrades to omethoate (OMT) and both disappear in about two months. Therefore, simple, sensitive, cost-effective and accurate methods for the determination of dimethoate, possibly suitable for in-field application, can be of great interest. In this work, a quick screening method, possibly useful for organic cultures certification will be presented. DMT and OMT in water and on olive leaves have been detected by surface enhanced Raman spectroscopy (SERS) using portable instrumentations. On leaves, the SERS signals were measured with a reasonably good S/N ratio, allowing us to detect DMT at a concentration up to two orders of magnitude lower than the one usually recommended for in-field treatments. Moreover, detailed information on the DMT distribution on the leaves has been obtained by Raman line- (or area-) scanning experiments.

Surface Engineering of Gold Nanorods for Cytochrome c Bioconjugation: An Effective Strategy To Preserve the Protein Structure.

The surface of gold nanorods (Au NRs) has been appropriately engineered to achieve a suitable interface for bioconjugation with horse heart cytochrome c (HCc). HCc, an extensively studied and well-characterized protein, represents an ideal model for nanoparticle (NP)-protein conjugation studies because of its small size, high stability, and commercial availability. Here, the native state of the protein has been demonstrated for the first time, by means of Raman spectroscopy, to be retained upon conjugation with the anisotropic Au nanostructures, thus validating the proposed protocol as specifically suited to mostly preserve the plasmonic properties of the NRs and to retain the structure of the protein. The successful creation of such bioconjugates with the retention of the protein structure and function along with the preservation of the NP properties represents a challenging but essential task, as it provides the only way to access functional hybrid systems with potential applications in biotechnology, medicine, and catalysis. In this perspective, the organic capping surrounding the Au NRs plays a key role, as it represents the functional interface for the conjugation step. Cetyltrimethylammonium bromide-coated Au NRs, prepared by using a seed-mediated synthetic route, have been wrapped with polyacrylic acid (PAA) by means of electrostatic interactions following a layer-by-layer approach. The resulting water-dispersible negatively charged AuNRs@PAA NPs have then been electrostatically bound to the positively charged HCc. The bioconjugation procedure has been thoroughly monitored by the combined analysis of UV-vis absorption, resonance Raman and Fourier transform infrared spectroscopies, transmission electron microscopy microscopy, and ζ-potential, which verified the successful conjugation of the protein to the nanorods.

Silver nanowires as infrared-active materials for surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is increasing in significance as a bioanalytical tool. Novel nanostructured metal substrates are required to improve performances and versatility of SERS spectroscopy. In particular, as biological tissues are relatively transparent in the infrared wavelength range, SERS-active materials suitable for infrared laser excitation are needed. Nanowires appear interesting in this respect as they show a very broad localized surface plasmon resonance band, ranging from near UV to near infrared wavelengths. The SERS activity of silver nanowires has been tested at three wavelengths and a fair enhancement at 1064 and 514 nm has been observed, whereas a very weak enhancement was present when exciting close to the nanowire extinction maximum. These experimentally measured optical properties have been contrasted with finite element method simulations. Furthermore, laser-induced optoacoustic spectroscopy measurements have shown that the extinction at 1064 nm is completely due to scattering. This result has an important implication that no heating occurs when silver nanowires are utilized as SERS-active substrates, thereby preventing possible thermal damage.

Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket.

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH- ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.

Structure-function relationships in human cytochrome c: The role of tyrosine 67.

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.