Fabrizio Bernini

pH-Dependent peroxidase activity of yeast cytochrome c and its triple mutant adsorbed on kaolinite.

The peroxidase activity of wild-type yeast cytochrome c and its triple mutant K72AK73AK79A adsorbed onto kaolinite was investigated as a function of pH and temperature. Both adsorbed proteins displayed an appreciable catalytic activity, which remained constant from pH 7 to pH 10, decreased below pH 7, and showed a remarkable increase at pH values lower than 4. In the whole pH range investigated the catalytic activity of the adsorbed wild-type cytochrome c was higher than that of the mutant. Both diffuse-reflectance UV-vis and resonance Raman spectroscopies applied on solid samples were used to probe the structural features responsible for the catalytic activity of the immobilized proteins. At neutral and alkaline pH values a six-coordinate low-spin form of cytochrome c was observed, while at pH < 7 the formation of a high-spin species occurred whose population increased at decreasing pH. The orientation and exposure of the heme to the substrate-strictly dependent on adsorption-was found to affect the peroxidase activity.

The Met80Ala point mutation enhances the peroxidase activity of immobilized cytochrome c

The effects of replacement of the axial methionine 80 heme ligand with a non-coordinating alanine on the peroxidase activity of kaolinite-immobilized cytochrome c were investigated at different pH values. The catalytic activity of the adsorbed mutant was found to be remarkably higher than that of wild-type cytochrome c. The pH dependence of Vmax and KM values is discussed in terms of accessibility of the substrates to the metal center and surface charge of kaolinite. Our approach, based on the combined use of adsorption on kaolinite and protein engineering, endows this bioinorganic interface with remarkable catalytic properties.

Enhancing Biocatalysis: The Case of Unfolded Cytochrome c Immobilized on Kaolinite

Cytochromes c (cytc) are heme proteins that are involved in several biological processes. The robust protein structure and the covalent attachment of the heme center to the polypeptide matrix allow (at least partial) protein folding and redox function even under hostile physicochemical conditions. The cytc family is among the most thoroughly investigated classes of electron-transfer (ET) proteins owing to its large availability and ease of purification. Moreover, these proteins can be easily engineered by site-directed mutagenesis. In the last decade, several studies have shown that non-native forms of cytc exhibit peroxidase activity (PA) toward several organic substrates including aromatic hydrocarbons, organosulfur compounds, and heterocycles. Given that enzymes are, in general, rather delicate molecules, as they are subjected to inactivation by temperature, pH, and ionic composition of the solution, cytc can be seen as valuable candidates for in vitro and/or industrial oxidative biocatalysis. It is known that cytc shows PA only when the “sixth” axial coordination site of the heme iron is vacant or when it bears a weak ligand.Interestingly, this catalytic property is induced by unfolding agents as a result of the cleavage or weakening of the native axial methionine bond to the iron and to the increase in solvent accessibility of the heme center caused by partial unfolding. 7] Therefore, the PA of cytc is directly related to the availability of the heme iron to undergo H2O2 binding and to the presence of a heme pocket that retains most of its structural features for substrate binding. Moreover, adsorption of cytc on some inorganic surfaces has been found to induce PA. 8–11] This would open the way to the production of a heterogeneous catalyst whose efficiency depends on the surface area; moreover, the use of such a catalyst would facilitate separation and recovery of the reaction products. The possibility of using supported cytc as a heterogeneous catalyst is intriguing because this protein is known to be very versatile and robust and can be easily mutated to work under a wide range of conditions. Therefore, exploitation in bioand environmental catalysis is at hand. We previously investigated the thermodynamic aspects of the immobilization of cytc and some variants on kaolinite (Nak); the variants were obtained by replacing the surface lysine residues in the 72-, 73-, and 79-positions with alanine residues. Cytc is able to strongly and quantitatively adsorb onto the surface of Nak through electrostatic interactions. This bioinorganic interface was found to exhibit appreciable catalytic activity in the oxidation of guaiacol (Gc) to tetraguaiacol (tetraGc) in the presence of H2O2. [6] The aim of this paper is to find conditions under which the catalytic properties are amplified and improved. Protein unfolding is known to increase the PA of cytc, but immobilization on an anionic surface increases conformational stability. 8, 9] For these reasons, we chose to study the effects of the well-known unfolding agent urea, not only on the native protein but also on selected variants. The choice of Lys-to-Ala mutants at the 72-, 73-, and 79-positions was made for the following reasons: 1) These lysine residues are key contributors to the positively charged domain of cytc involved in the binding to Nak. Therefore, these residues may influence the adsorption geometry and, consequently, may affect the PA of immobilized cytc. 2) These lysine residues are placed near the crevice exposing the heme to the solvent and, therefore, could control access of a substrate to the heme center. 3) These lysine residues, and in particular Lys79, are located near the foldon, which is first involved in urea-induced unfolding. The PA was spectrophotometrically determined by following the oxidation of Gc to tetraGc due to H2O2 (Supporting Information S3):

Effective and Selective Trapping of Volatile Organic Sulfur Derivatives by Montmorillonite Intercalated with a μ-oxo Fe(III)–Phenanthroline Complex

The μ-oxo Fe(III)-phenanthroline complex [(OH2)3(Phen)FeOFe(Phen) (OH2)3]+4 intercalated in montmorillonite provides a stable hybrid material. In this study, the ability and efficiency of this material to immobilize thiols in gas phase, acting as a trap at the solid-gas interface, were investigated. Aliphatic thiols containing both hydrophilic and hydrophobic end groups were chosen to test the selectivity of this gas trap. DR-UV-vis, IR, elemental analysis, thermal analysis and evolved gas mass spectrometry, X-ray powder diffraction, and X-ray absorption spectroscopy techniques were employed to characterize the hybrid material before and after thiol exposure and to provide information on the entrapping process. Thiol immobilization is very large, up to 21% w/w for heptanethiol. In addition, evidence was obtained that immobilization occurs through the formation of a covalent bond between the iron of the complex and the sulfur of the thiol. This provides an immobilization process characterized by a higher stability with respect to the methods based on physi-adsorption. Thiol immobilization resulted thermally reversible at least for 20 adsorption/desorption cycles. Unlike standard desulfurization processes like hydrotreating and catalytic oxidation which work at high temperatures and pressures, the present system is able to efficiently trap thiols at room temperature and pressure, thus saving energy. Furthermore, we found that the selectivity of thiol immobilization can be tuned acting on the amount of complex intercalated in montmorillonite. In particular, montmorillonite semisaturated with the complex captures both hydrophobic and hydrophilic thiols, while the saturated montmorillonite shows a strong selectivity toward the hydrophobic molecules.

Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity.

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Abstract The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.