Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/9531
Title: New insights into heme peroxisases: internediates and mechanisms
Authors: Gumiero, Andrea
Supervisors: Raven, Emma
Moody, Peter
Award date: 1-Jul-2011
Presented at: University of Leicester
Abstract: 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.
Links: http://hdl.handle.net/2381/9531
Type: Thesis
Level: Doctoral
Qualification: PhD
Sponsors / Funders: Leverhulme trust
Appears in Collections:Theses, Dept. of Chemistry
Leicester Theses

Files in This Item:
File Description SizeFormat 
Andrea Gumiero Thesis.pdf16.32 MBAdobe PDFView/Open


Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.