Research

Our research is in the area of biophysical chemistry.
The research, supported by EU and national funding, is carried out in collaboration with various leading Italian, European and North-American groups. collaborations
The main activity and expertise of the team are directed towards the spectroscopic characterization of heme proteins. All the proteins contain the protoporphyrin IX prosthetic group and conserved aminoacid residues considered to be important for the catalytic activity. Spectroscopy has played a fundamental role in structure-activity studies. In particular the combined analysis of electronic absorption (UV-Vis), resonance Raman (RR), and FT-IR spectroscopies of native and mutant enzymes, at different pH and temperature, in the presence of various exogenous ligands, in solution and single crystal forms, enable the role of the key residues in the heme cavity to be defined and their structure-function properties to be elucidated.

The combination of resonance Raman and electronic absorption spectroscopy, especially when applied to a study incorporating native, recombinant and mutant proteins of key residues, give detailed information at the molecular level of the active site.

Considerable information has been obtained on:

the Fe coordination state, the stability of the protein architecture for various heme proteins,

the roles of the conserved residues in the heme cavity of peroxidases,

the hydrogen bonding network which links the proximal and distal sides of the heme in peroxidases,

the factors influencing the stabilization of exogenous ligands (OH-, F-, CO) and their spectroscopic markers,

the degree of conjugation of the vinyl substituents with the porphyrin macrocycle.
Ongoing  research projects include

A)  Spectroscopic investigation of heme containing peroxidases in solution. Study of the effect of pH, temperature and mutation on the stability. Structure-function relationship.

  1. Human eosinophil peroxidase (EPO): a detailed spectroscopic study is performed on EPO in order to obtain information on the origin of its anomalous RR spectra (in collaboration with the Chemistry Dept, Mc Gill University, Montreal, Canada.)
  2. Eukaryotic extracellular catalase-peroxidase. A detailed resonance Raman and electronic absorption investigation has been carried out on Katg from Magnaporthegrisea (in collaboration with the Institute of Chemistry, University of Agricultural Sciences, Vienna, Austria.).

B) Spectroscopic investigation of globins in solution and single crystals. Characterization of the ferrous and ferric forms and the compelxes with exogeneous ligands  (CO, fluoride, H2S, hydroxo, Cyanide)to understand the structure-activity relationships of truncated hemoglobins (trHbs) from cold-adapted bacteria in comparison with their thermophylic orthologues

  1. The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida Fusca and a combinatorial set of mutants, in each of which the distal TrpG8, TyrB10, and TyrCD1 amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound ligands. This team will characterize the bacterial hemoglobins by optical spectroscopy (UV-visible and infrared absorption, Raman and resonance Raman scattering), and EPR under chemically and physically varied conditions. The scope is to to obtain information on the interactions of the heme with the surrounding aminoacids and in the presence of exogenous ligands and various substrates (in collaboration with the University "La Sapienza", Roma, Italy, and Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires,Argentina .
  2. The structural and functional properties of the  heme pocket of the 2/2 Hemoglobin of Cold Adapted Pseudoalteromonas haloplanktis TAC125, using UV-Vis, RR, and EPR spectroscopies, and selected distal mutants (TyrB10Phe and TyrCD1Phe). This work represents the first detailed characterization of a cold-adapted bacterial Hb in order to study the effect of adaptation on cold environment.In collaboration with Institute of Protein Biochemistry, CNR, 80131 Naples, Italy and Departamento de Quimica Inorganica, Analitica y Quimica Fisica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires,Argentina.
    3.
C) Surface Enhanced Resonance Raman Spectroscopy for the detection of residuals of chemicals in breakfast cereal.
    Benzophenone (BP) and 4-methylbenzophenone (4MBP) are commonly used in food packaging. Due to their volatility they can migrate through the packaging into the food. Recently BP and 4MBP has been found in cereal products up to 678 ppm. In the present work, a chemical sensor for the quantitative detection of BP and 4MBP extracted from spiked cereal samples has been developed using Surface Enhanced Raman Spectroscopy on Ag nanoparticles functionalized with the molecular assembler bis-acridinium dication lucigenin. (This research project has been funded by the Tuscany Region: Programma per la Ricerca Regionale in Materia di Salute, n. 273).






     
     


     



     
     

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