Despite the numerous studies on the adsorption of different proteins onto mesoporous titanium dioxide and indications on the important role of buffer solutions in bioactivity, a systematic study on the impact of the buffer on the protein incorporation into porous substrates is still lacking. We here studied the interaction between a commercial mesoporous TiO and three of the most used buffers for protein incorporation, i.e. HEPES, Tris and phosphate buffer. In addition, this paper analyzes the adsorption of horse heart myoglobin (hhMb) onto commercial mesoporous TiO as a model system to test the influence of buffers on the protein incorporation behavior in mesoporous TiO. N sorption analysis, FT-IR and TGA/DTG measurements were used to evaluate the interaction between the buffers and the TiO surface, and the effect of such an interaction on hhMb adsorption. Cyclic voltammetry (CV) and electron paramagnetic resonance (EPR) were used to detect changes in the microenvironment surrounding the heme. The three buffers show a completely different interaction with the TiO surface, which drastically affects the adsorption of myoglobin as well as its structure and electrochemical activity. Therefore, special attention is required while choosing the buffer medium to avoid misguided evaluation of protein adsorption on mesoporous TiO.

The cytoglobins of the Antarctic fish Chaenocephalus aceratus and Dissostichus mawsoni have many features in common with human cytoglobin. These cytoglobins are heme proteins in which the ferric and ferrous forms have a characteristic hexacoordination of the heme iron, i.e. axial ligation of two endogenous histidine residues, as confirmed by electron paramagnetic resonance, resonance Raman and optical absorption spectroscopy. The combined spectroscopic analysis revealed only small variations in the heme-pocket structure, in line with the small variations observed for the redox potential. Nevertheless, some striking differences were also discovered. Resonance Raman spectroscopy showed that the stabilization of an exogenous heme ligand, such as CO, occurs differently in human cytoglobin in comparison with Antarctic fish cytoglobins. Furthermore, while it has been extensively reported that human cytoglobin is essentially monomeric and can form an intramolecular disulfide bridge that can influence the ligand binding kinetics, 3D modeling of the Antarctic fish cytoglobins indicates that the cysteine residues are too far apart to form such an intramolecular bridge. Moreover, gel filtration and mass spectrometry reveal the occurrence of non-covalent multimers (up to pentamers) in the Antarctic fish cytoglobins that are formed at low concentrations. Stabilization of these oligomers by disulfide-bridge formation is possible, but not essential. If intermolecular disulfide bridges are formed, they influence the heme-pocket structure, as is shown by EPR measurements.

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Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

Antioxidants and Redox SignalingStructural and functional characterization of the globin-coupled sensors of Azotobacter vinelandii and Bordetella pertussis

Despite the intensive research on protein adsorption in mesoporous materials, the effect of (de)­hydration and confinement on the adsorbed protein’s stability and activity is poorly understood. In this paper, we study the effect of differences in structural features (pore size) and drying time on the adsorption and structural stability of horse heart myoglobin (hhMb) on mesoporous titanium dioxide. Infrared spectroscopy (DRIFT) and thermal analysis (TGA) coupled to a quadrupole mass spectrometer (TGA–MS) were used to evaluate the impact of the confinement in different pores and hydration on the myoglobin secondary structure. Electron paramagnetic spectroscopy (EPR) was applied to identify the changes in the heme and its close surrounding. The peroxidase-like activity of myoglobin toward 2,2′-azinobis­(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide allowed us to detect changes in the protein activity after adsorption in pores with different sizes and drying for different periods of time. The results show a clear effect of the pore size and drying time on the secondary structure of hhMb, which is confirmed by differences induced in the catalytic activity of the adsorbed proteins. Therefore, we recommend evaluating the effect of both hydration and confinement in future applications involving biomolecule adsorption in porous matrices.

A series of ZnTi layered double hydroxides (LDH) with different Zn/Ti ratios are prepared and used as catalysts for photodegradation of salicylic acid (SA) under visible light. The catalysts are characterized by X-Ray diffraction, diffuse reflectance infrared Fourier transform spectroscopy, UV-vis diffuse reflectance spectroscopy, thermogravimetry, electron paramagnetic resonance and N2 adsorption-desorption. The results show that SA anions bind to the LDH surface and that an electron can be excited from the HOMO in the adsorbed molecules to the conduction band of the LDH under visible light illumination. This charge transfer further leads to an effective photodegradation and mineralization of SA with better conversion results than on P25 titania.Key factors influencing the charge-transfer process in LDH are the high surface area and the Ti/Zn ratio of the LDH materials. The combination of highly dispersed Zn 2+ and Ti 4+ cations in the brucite-like sheets of the LDH allows for a better charge separation, which also accounts for the high photocatalytic activity. The present results show that superoxide radicals play a role in the visible-light induced degradation of SA on LDH, while no  OH radicals are formed. In contrast to LDH, the light-induced degradation pathway of SA over P25 titania leads to the formation of CO2 -, a relatively stable anion that may hamper further conversion to CO2 and hence limit the photocatalytic performance. The introduction of an electron acceptor, such as peroxydisulfate, further improves the degradation and mineralization of SA over LDH, but care should be taken not to use an electron acceptor that can easily adsorb to the LDH surface, such as H2O2. ZnTi LDH are thus very promising alternatives to TiO2 for the photodegradation of colorless organic pollutants, such as SA, under visible light irradiation. Highlights-ZnTi layered double hydroxides (LDH) allow mineralization of salicylic acid under visible light -Visible light induces charge transfer from the adsorbed molecule to the semiconductor -The Ti/Zn ratio and specific surface area of ZnTi LDH influence the photoactivity -Photodegradation of SA involves different radical intermediates in LDH than in titania

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