Structure function relationships in catalase peroxidases

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.

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. Because of these unique enzymatic features these oxidoreductases are excellent model oxidoreductases to study and understand how the protein matrix and the design of the active site and of the substrate channel modulates one- and two-electron transfer pathways and chemical reactivity. In the last years the crystal structures of four catalase-peroxidases were published showing unique KatG-typical post-translational modifications and giving the framework to elucidate structure-function relationships in this project. An extensive search for KatG encoding genes in all available databases revealed the existence of more than 300 sequences, most of them in bacterial genomes, but some in eukaryotic organisms like protists and fungi. Chaotic distribution among species and incongruous phylogenetic construction indicated existence of numerous lateral gene transfers in addition to duplication events and regular speciation. Based on this analysis one cyanobacterial KatG from Synechocystis as well as one fungal KatG from Magnaporthe grisea were chosen for functional and mechanistic studies. Upon using gas chromatography coupled with mass spectrometry and peroxide labeling, it was demonstrated that O2 in KatG turnover is formed by two-electron oxidation without breaking the O-O bond in H2 O2 . Also active site variants with very low catalase but normal or even enhanced peroxidase activity were shown to oxidize H2 O2 by a non-scrambling mechanism similar to monofunctional heme catalases. Spectroelectrochemical investigations showed that the existence of the KatG-typical distal side adduct Trp-Tyr- Met, which is essential for catalase but not peroxidase activity, has no impact on the actual redox chemistry of the heme iron. The reduction potential of wild-type KatG was found to be -226 mV (Synechocystis) or -186 mV (Magnaporthe grisea KatG1). The reduction potentials of the KatG variants without the Trp-Tyr-Met adduct (Y249F) were very similar. Comprehensive rapid kinetic and spectral analysis of the reactions of KatGs from three different sources (Synechocystis PCC 6803, Burkholderia pseudomallei and Mycobacterium tuberculosis) with peroxoacetic acid and hydrogen peroxide revealed that, independent of KatG, but dependent on pH, two low-spin forms dominate in the catalase cycle. By contrast, oxidation of KatGs with peroxoacetic acid resulted in intermediates with different spectral features that also differed among the three KatGs. These discrepancies were reflected also by high-field EPR spectroscopical studies that allowed analysis of the electronic features of the involved redox intermediates, including formation, localization and quenching of protein radicals.