Pulsed EPR of dynamics of catalytic residues in heme enzymes

Heme enzymes conduct a myriad of diverse biological functions such as dioxygen transport, storage and reduction, electron transport and oxidation of manifold organic and inorganic compounds. Understanding the mechanisms behind these reactions needs the elucidation of the (electronic) structure of the relevant redox intermediates and of the role of (catalytic) amino acids in the heme cavity. Very often active site residues are mobile but the relationship between distinct conformations and functionality in most cases is unclear. This project, for the first time, focuses on the elucidation of the role of mobile arginines in the model heme enzymes chlorite dismutase (Cld) and catalase-peroxidase (KatG) in ligand or substrate binding and conversion, modulation of heme reactivity as well as stability and interconversion of redox intermediates by means of advanced electron paramagnetic resonance (EPR) spectroscopy, namely pulsed EPR techniques such as ENDOR and HYSCORE. The recombinant production of both wild-type and mutant Clds and KatGs is well established in the laboratory of the co-applicant and the proteins will be probed under distinct conditions like pH, ionic strength, spin-, coordination and oxidation states as well as under distinct isotopic labelling (amino acids and/or prosthetic group) regimes. Pulsed ENDOR and HYSCORE are complementary techniques for the determination of the interaction of the unpaired electron with surrounding nuclei with a non-zero spin. So far there are only a few examples of the application of these methods in the biophysical analysis of heme enzymes. In the present work the heme iron will be chosen as the observer position and the interaction with the surrounding nitrogen atoms will be detected. In case of Arg, it is the distal guanidinium residue in KatG and Cld, which is either pointing ‘in’ or ‘out’ and has two possible nitrogen atoms for interaction with the unpaired electron(s) of the Fe-atom. Due to the presence of several other nitrogens originating from the heme and imidazol groups in the close proximity of the Fe-atom, 14N/15N isotope exchange is planned to simplify the spectra and distinguish between the different nitrogen sources. Furthermore, high frequency ENDOR (95 GHz) will be considered if necessary to separate 14N/15N frequencies. In catalase-peroxidase, there is a second mobile Arg ~20 Å away from the heme centre. Conformational changes of this Arg residue have an electron pushing or abstracting effect on the heme pocket and this might be visible via the electronic structure of the KatG-typical Trp-Tyr-Met adduct located between the Arg and the prosthetic group. Alternatively to the heme iron, a transiently formed Tyr-radical on the peculiar KatG-typical Trp-Tyr-Met adduct could serve as observer position for pulsed ENDOR and HYSCORE. Summing up, this project aims at the evaluation of the power of pulsed EPR techniques in the elucidation of structure-function relationships of metalloenzymes. It will give new insights into the dynamics of catalytic Arg in two distinct heme oxidoreductases.