Structure-function relationship in catalase-peroxidase

In developing ideas of how protein structure modifies heme reactivity, class I peroxidases are an exciting field of research. Despite striking sequence homologies, class I members (yeast cytochrome c peroxidase, ascorbate peroxidase and catalase-peroxidases) exhibit dramatic differences in catalytic activity and substrate specificity. Cytochrome c peroxidase (CCP) is unusual in that it uses another protein (cytochrome c) as a redox partner, whereas ascorbate peroxidases (APXs) prefer the anion ascorbate as electron donor. Both CCP and APX do not exhibit substantial catalase activity. By contrast, multifunctional catalase-peroxidases (KatGs) exhibit a catalase activity comparable to monofunctional catalases in addition to function as peroxidase of broad-specificity. But the structural basis for this extraordinary reactivity is unknown. In order to elucidate structure-function relationships of catalase-peroxidases, in this project we plan to perform comprehensive mutational analysis in combination with kinetic and spectroscopic (UV-Vis, resonance Raman and electronic paramagnetic) investigations. Three recombinant catalase-peroxidases will be investigated in this project. Since the three-dimensional structure of KatG is unknown crystallization studies of recombinant holoproteins and truncated proteins which lack parts at the N- and C-terminal region as well as parts of the KatG-typical flexible loops are planned. Based on multiple sequence alignment and secondary structure prediction, mutational analysis will be performed. Besides manipulations at the highly conserved active site, the project is focused on KatG specific insertions, three of them are typical for KatGs showing highly conserved sequence patterns. A hypothesis for the roles of these KatG specific insertions is presented and (based on data from molecular modeling in combination with site-direted fluorescence labeling) site-directed mutagenesis experiments are planned and recombinant variants are produced. The spectral and kinetic features of the variants will be investigated by steady-state and transient-state kinetic measurements. Information about iron coordination and spin states, hydrogen bonding networks in ferric and ferrous proteins, the nature (e.g. existence, location and role of protein radicals) and reactivity of the redox intermediates as well as the influence of the protein matrix on heme substituent orientation and heme side residue geometries and distances will be investigated by resonance Raman and EPR spectroscopy. In the proposal a hypothesis for the possible electronic structure of KatG intermediates is presented and should be tested within this project.

In developing ideas of how protein structure modifies heme reactivity, class I peroxidases are an exciting field of research. Despite striking sequence homologies, class I members (yeast cytochrome c peroxidase, ascorbate peroxidase and catalase-peroxidases) exhibit dramatic differences in catalytic activity and substrate specificity. Cytochrome c peroxidase (CCP) is unusual in that it uses another protein (cytochrome c) as a redox partner, whereas ascorbate peroxidases (APXs) prefer the anion ascorbate as electron donor. Both CCP and APX do not exhibit substantial catalase activity. By contrast, multifunctional catalase-peroxidases (KatGs) exhibit a catalase activity comparable to monofunctional catalases in addition to function as peroxidase of broad-specificity. But the structural basis for this extraordinary reactivity is unknown. In order to elucidate structure-function relationships of catalase-peroxidases, in this project more than 35 mutants were designed and analysed by a combination of steady- and presteady-state kinetic and spectroscopic (UV-Vis, resonance Raman and electronic paramagnetic, circular dichroism) investigations. It has been shown that KatGs exhibit several structural peculiarities (e.g. insertions not found in other heme peroxidases or an extraordinary covalent adduct at the distal heme site), which are essential for the catalatic activity (i.e. the hydrogen peroxide oxidation reaction) but not the peroxidase activity. The project was successful in characterizing the spectral and electronic features of the redox intermediates compounds I, II and III as well as in the elucidation and localization of protein (tryptophanyl- and tyrosyl-) radicals. It was demonstrated how the hydrogen bond network of KatG is responsible for (i) the formation and stability of protein radicals, (ii) the stability of the heme cavity and (iii) the guiding, binding and oxidation of H2 O2 to molecular oxygen. A mechanism for the catalase activity was proposed, which is completely different from that known from monofunctional catalases. It involves the formation of an alternative compound I, which has a high reactivity with the second hydrogen peroxide molecule. Mechanistically, the reaction of this compound I with H2 O2 would be similar to that of an oxyferryl compound II with H2 O2 . The reaction product would be a ferrous- dioxy/ferric-superoxide complex containing a protein radical site. Storing one-oxidation equivalent could guarantee that (i) finally dioxygen and not superoxide is released when ferric KatG is formed, and (ii) that the dioxygen adduct is much more unstable compared to the typical oxymyoglobin-like compound III. As a consequence this species does not accumulate and the enzyme cycles rapidly.