New insights into heme peroxisases: internediates and mechanisms

Heme peroxidises catalyse the H2O2-dependent oxidation of substrates in a two-step process, through formation of two oxy-ferryl intermediates known as Compound I and Compound II. Despite the considerable effort worldwide, important aspects about the reactivity of these enzymes are still to be clarified. Amongst all, the determination of the nature of the Fe-O bond in the oxy-ferryl intermediates, as well as the mechanism by which protons are delivered to the oxy-ferryl species during turnover, are of highest relevance. In this thesis, high resolution crystal structures of both Compound I and Compound II intermediates in two heme peroxidases, cytochrome c peroxidise (CcP) and ascorbate peroxidise (APX), are presented. In order to rule out the photoreduction arising from X-ray exposure during data collection, which causes alteration of ferryl intermediate structures, a multicrystal method has been employed. Results indicate that Compound I, with an Fe-O distance of 1.63 Å for CcP and 1.73 Å for APX, is consistent with an unprotonated oxy-ferryl species (FeIV=O), whereas Compound II, with an Fe-O bond length of 1.83 Å and 1.84 Å for CcP and APX respectively, is consistent with a protonated oxy-ferryl species (FeIV-­‐OH). Also presented in this thesis is the 2.40 Å structure of resting ferric CcP at room temperature obtained, for the first time, by neutron crystallography. This study allowed to establish the location of individual, exchangeable hydrogen atoms thus revealing the protonation states of several key active site residues in the distal (Arg48, Trp51, His52) and proximal (His163, Trp191, Asp235) heme regions. This information was used to revise the reaction mechanism of heme peroxidises and also to infer a possible delivery pathway of protons during turnover. All together, these data not only clarify long-standing inconsistencies on the nature of the oxy-ferryl species, but they also provide new insights into The reaction mechanism of heme peroxidises and provide important information which May apply to other categories of heme enzymes such as the cytochromes P450 and NO synthases

Gas-in-Liquid Foam Templating as a Method for the Production of Highly Porous Scaffolds

In the present work, a novel synthetic methodology for the preparation of scaffold of biopolymeric nature is described. In particular, a porous gelatin scaffold was prepared by foam templating. The gas phase, nitrogen, was generated by means of the reaction between sulfamic acid and sodium nitrite in situ a concentrated solution of gelatin and in the presence of a suitable polymeric surfactant in association with sodium dodecyl sulfate. The foam was prepared at a temperature of 45 °C and then let gel at 5 °C. After purification, the physical gel was auto-cross-linked with EDC and freeze-dried. The scaffold synthesized with this technique presents a morphology characterized by voids of spherical symmetry highly interconnected by a plurality of interconnects, and, as a consequence, is particularly suited for cell culturing. In more quantitative terms, voids and interconnects are characterized by an average diameter of 230 and 90 μm, respectively. Preliminary tests of cell culturing demonstrated the suitability of such a scaffold for tissue engineering applications

Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H2O and D2O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes

Vegetation, soil and hydrology management influence denitrification activity and the composition of nirK-type denitrifier communities in a newly afforested riparian buffer

Soil microbial community composition and activity could be affected by suitable manipulation of the environment they live in. If correctly applied such an approach could become a very effective way to remediate excess of chemicals. The concentration of nitrogen, especially nitrate deriving from agricultural managements, is generally found to increase in water flow. Therefore, by forcing the water flow through a buffer strip specifically designed and possibly afforested with suitable plant species, may result effective in reducing high nitrogen contents. The management of a riparian buffer may definitely affect the soil microbial activities, including denitrification, as well as the composition of the community. The present study reports on the changes occurred in terms of denitrifying microbial community composition, as compared to that of a neighbouring agricultural area, as a consequence of hydraulic management coupled to the suspension of farming practices and to the development of the woody and herbaceous vegetation. With this aim, denitrification was repeatedly measured and the data obtained were related to those deriving from a specific analysis of bacterial groups involved in denitrification. nirK, encoding for nitrite reductase, an enzyme essential for the conversion of nitrite to nitric oxide and considered the key step in the denitrification process, was chosen as the target gene. The main results obtained indicated that denitrification activity changes in riparian buffer as compared to agricultural soil and it is strongly influenced by carbon availability and soil depth. Although no significant differences on the community composition between superficial (0\u201315 cm) and medium (40\u201355 cm) layers were observed, the nirK-type denitrifier community was shown to significantly differ between riparian and agricultural soils in both surface and medium layers

Six-Minute Walking Distance Improvement after Pulmonary Rehabilitation Is Associated with Baseline Lung Function in Complex COPD Patients: A Retrospective Study

INTRODUCTION: Conflicting results have been so far reported about baseline lung function, as predicting factor of pulmonary rehabilitation (PR) efficacy. AIM: To ascertain whether or not baseline lung function could predict a benefit in terms of a significant change in 6-min walk test (6 MWT) after PR. METHODS: Seventy-five stable moderate-to-severe COPD inpatients with comorbidities (complex COPD), allocated to a three-week PR program, were retrospectively evaluated. Pulmonary function, 6 MWT, dyspnea (BDI/TDI), and quality of life (EQ-VAS) were assessed before and after PR program. The patients were divided into two groups depending on the change in 6 MWT (responders > 30 m and nonresponders 64 30 m). Logistic regression analysis was used. Results. After PR, 6 MWT performance all outcome measures significantly improved (P < 0.01). Compared to nonresponders (N = 38), the responders (N = 37) had lower values in baseline lung function (P < 0.01). Logistic regression analysis showed that FEV1 < 50% pred and TL, CO < 50% pred were independent predictors of PR efficacy. CONCLUSIONS: Our study shows that in stable moderate-to-severe complex COPD inpatients, baseline lung function may predict the response to PR in terms of 6 MWT. We also found that complex COPD patients with poor lung function get more benefit from PR