Distribution of H$$^\upbeta$$ Hyperfine Couplings in a Tyrosyl Radical Revealed by 263 GHz ENDOR Spectroscopy

Abstract$$^1$$ 1 H ENDOR spectra of tyrosyl radicals (Y$$^\bullet$$ ∙ ) have been the subject of numerous EPR spectroscopic studies due to their importance in biology. Nevertheless, assignment of all internal $$^1$$ 1 H hyperfine couplings has been challenging because of substantial spectral overlap. Recently, using 263 GHz ENDOR in conjunction with statistical analysis, we could identify the signature of the H$$^{\upbeta _2}$$ β 2 coupling in the essential Y$$_{122}$$ 122 radical of Escherichia coli ribonucleotide reductase, and modeled it with a distribution of radical conformations. Here, we demonstrate that this analysis can be extended to the full-width $$^1$$ 1 H ENDOR spectra that contain the larger H$$^{\upbeta _1}$$ β 1 coupling. The H$$^{\upbeta _2}$$ β 2 and H$$^{\upbeta _1}$$ β 1 couplings are related to each other through the ring dihedral and report on the amino acid conformation. The 263 GHz ENDOR data, acquired in batches instead of averaging, and data processing by a new “drift model” allow reconstructing the ENDOR spectra with statistically meaningful confidence intervals and separating them from baseline distortions. Spectral simulations using a distribution of ring dihedral angles confirm the presence of a conformational distribution, consistent with the previous analysis of the H$$^{\upbeta _2}$$ β 2 coupling. The analysis was corroborated by 94 GHz $$^2$$ 2 H ENDOR of deuterated Y$$_{122}^\bullet$$ 122 ∙ . These studies provide a starting point to investigate low populated states of tyrosyl radicals in greater detail.

Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.