Structure-function relationships in catalase-peroxidases
- Biotechnologie
- Forschungscluster "Bioindustrielle Technologien"
Abstract
Catalase-peroxidases (KatGs) are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good models to study and understand the role of electron pathways both in monofunctional catalases and peroxidases. In the last three years the crystal structures of four catalase-peroxidases were published showing unique KatG-typical features and giving now the framework to elucidate structure-function relationships and design experiments on a rational basis. Based on already successfully performed investigations on KatG from Synechocystis PCC 6803 in our group, in the present proposal a powerful combination of site-directed mutagenesis, multimixing UV-Vis- and CD-stopped-flow spectroscopy, multifrequency freeze-quench EPR spectroscopy in combination with isotopic labeling of tryptophan and tyrosine residues, resonance Raman spectroscopy and X-ray crystallography will be used to provide answers to the following still unsolved problems, finally leading to be a better understanding which structural features govern the electronic pathways and redox reactions observed in multifunctional KatGs. In detail, it is the aim of the present work to understand the electronic absorption and EPR spectra of the redox intermediates ferrous KatG, ferric KatG, compound I, compound II and compound III. Previous work clearly indicated that (i) depending on reaction conditions (pH, nature of peroxide) electronic isomers can be formed exhibiting distinct spectral features, and (ii) that at least two distinct protein radicals (Trp· and Tyr·) contribute to them. The kinetics of interconversion in the absence and presence of one- and two-electron donors between this redox intermediates will be investigated. A proposed hypothesis regarding the mechanism of hydrogen peroxide reduction and oxidation (i.e. the KatG-typical catalatic activity), which is completely different to the actual widespread model, will be tested. Finally, it is intended to probe the role of potential substrate channels in the bifunctional catalysis of KatG. Binding sites for (the still unknown) peroxidase substrate(s) will be tested. Upon combining these data with those obtained by the kinetic and spectroscopic experiments a better understanding of the bifunctional activity of KatGs in particular as well as of one- and two-elcetron pathways in heme peroxidases in general will be obtained.
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Project staff
Christian Obinger
Univ.Prof. Mag.rer.nat. Dr.rer.nat. Christian Obinger
christian.obinger@boku.ac.at
Tel: +43 1 47654-10011
Project Leader
01.01.2006 - 31.12.2008
Christa Jakopitsch
Mag.Dr. Christa Jakopitsch
Tel: +43 1 47654-33061
Sub Projectleader
01.01.2006 - 31.12.2008
BOKU partners
Funder
