Interrogating peroxidase intermediate I : a rapid freeze quench \u2013 EPR approach

Abstract: This work is focused on the in-depth spectroscopic analysis of horseradish peroxidase (HRP) and chloroperoxidase (CPO), proteins selected for their scientific and industrial relevance. HRP, being extensively employed in immunoassays, has a crucial biochemical role, while CPO's structural and functional resemblance to cytochrome P450 emphasizes its importance in pharmacology. The enzymatic cycle of both peroxidases goes via an oxidized intermediate state, called Compound I. It stores two oxidizing equivalents, one in an iron(IV)-oxo moiety, and one as a free radical on the porphyrin ring or on an amino acid in the protein. Its investigation forms a central aspect of this research. Although abundantly studied, the exact description of the difference in the electronic structure of Compound I of various heme proteins remains challenging. The employed methodologies consist of established techniques, notably Continuous-Wave and Pulsed Electron Paramagnetic Resonance (EPR), UV-Vis spectroscopy, and stopped-flow spectrophotometry, alongside novel methods devised by the author. UV-Vis spectroscopy and stopped-flow spectrophotometry serve as standard tools in biotechnology, allowing the basic characterization and reaction kinetics of enzyme resting states and intermediates involved in enzyme turnover. Pulsed EPR, utilised in an attempt to elucidate the electron spin distribution of the resting state and Compound I forms of HRP and CPO, offers advantages in examining hyperfine interaction between unpaired electron(s) and surrounding nuclei. With the intention of enhance CPO production, an innovative solid substrate method has been explored, promising potential for scalability. Caldariomyces fumago-derived CPO production presents specific challenges due to cultivation and purification demands. Yet, the solid substrate method offers advantages, including increased yields, higher purity and cost-effectiveness, crucial for industrial applications. Furthermore, the author made crucial contributions to the developmental stages of three different rapid freeze quench apparatus used for trapping Compound I from CPO immediately after the initiation of the catalytic cycle. This involvement spanned from mechanical and technical creation to test, improvement and calibration of all three systems. EPR is a fundamental tool in exploring the electronic state of paramagnetic centers in proteins, such as the resting state and Compound I in HRP and CPO. M. Bracci ix Pulsed EPR techniques, such as HYSCORE and ENDOR, were utilized to investigate the resting state of both enzymes, providing insights into the electronic structure of their active centers and highlight their differences. To aid in the interpretation of HYSCORE spectra, a user-friendly graphical interface was developed, based on MATLAB and EasySpin, enabling researchers to manipulate experimental data and conduct simulations, even with minimal coding expertise. A theoretical model of the electronic structure of Compound I, previously proposed and based on the crystal field theory, was revisited. Starting from the energy of the t2g levels of Fe(IV) and a model for the magnetic interaction between the ion and the radical, this theory is suitable for interpreting the features of continuous wave EPR spectra, as demonstrated by the tests performed to literature data on the Compound I of different heme proteins. Subsequently, it was effectively used to simulate the CW EPR spectra of the intermediate of the proteins under investigation in this study. Finally, HYSCORE and ENDOR of the Compound I form of HRP and CPO revealed crucial differences in their 14N and 1H hyperfine couplings, which could be related to the difference in location of the radical