Quantitative Investigation Of The Structure And Function In Peptide Deformylase Studied By X-ray Absorption Spectroscopy

Metalloprotein is a generic term referring to a protein that contains a metal ion cofactor. Almost40%of the proteins of the Protein Data Bank are members of this category. Metalloproteins are involved in a wide range of important biological processes and fundamental chemical reactions. Chemical processes and reactions involving metalloproteins can be correlated to subtle structural changes around the metal ions that have to be precisely determined. X-ray absorption spectroscopy is a very powerful tool to determine the local structure around a metal ion in a biological system with subatomic-resolution. Moreover, samples can be studied in a variety of forms such as single protein crystals, frozen or room-temperature solutions and proteins inserted in membranes. This technique is the very suitable to describe the structure around the metal site in metalloproteins.In this thesis, we prepared a serial of PDF samples under different conditions and determined the local structure of their catalytic centers using the XANES analysis in the framework of the ab initio full Multiple Scattering theory. The main results are outlined in the following:1. We investigated the local structure of active sites in LiPDF andPDF in the native state. By using X-ray absorption near edge structure (XANES) we reconstructed successfully the local structures of their catalytic centers. Data allow to clarify the catalytic mechanism providing also the relevant structural information for applications of these proteins as specific drugs.2. LiPDF is strongly affected by changes of pH values. The enzymatic activity progressively decreases with pll up to7.0being almost constant from7.0up to11.0. We determined the local structure of the catalytic center by the MXAN code at different pH values and investigated both functional and structural changes of LiPDF. Results indicate that a correlation occurs between pH values and the enzymatic activity mainly due to the deprotonation of the coordinating water (watl) with the metal ion. Moreover, in the acid environment (low enzymatic activity), the zinc-watl-distance increases while the proton of watl localizes.3. We investigated the metal-dependent pH and enzymatic activity of local structures around the metal ion of PDFs. Results show that PDFs may exhibit a high enzymatic activity only when the nearest metal coordinating water molecule is placed at the distance of about2.10-2.55A with the metal ion while a low enzymatic activity is associated to metal-watl distances longer than2.55A or shorter than2.10A.