17O hyperfine spectroscopy to investigate water binding to organic radicals
by Fabian Hecker
Date of Examination:2022-05-18
Date of issue:2022-07-22
Advisor:Prof. Dr. Marina Bennati
Referee:Prof. Dr. Marina Bennati
Referee:Prof. Dr. Christian Griesinger
Persistent Address:
http://dx.doi.org/10.53846/goediss-9369
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Description:PhD Thesis
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Abstract
English
Hyperfine spectroscopy experiments detect nuclear spins around paramagnetic centers. They reveal magnetic interactions that contain valuable structural information on distance and orientation of the coupling partners. One can therefore use them, to study the magnetic nuclei in water molecules around organic radicals. Such radicals occur as important intermediates in enzymatic reactions.17O is a particularly interesting target nucleus to detect water molecules, since it does not exchange with other oxygen moieties in protein environments, allowing for unambiguous assignment of spectral signatures to H217O. It has already been used to study water coordination around transition metal ions. The low gyromagnetic ratio and high nuclear spin have, however, discouraged the use of 17O hyperfine spectroscopy to study water around organic radicals so far. This thesis shows the application of 17O hyperfine spectroscopy to organic nitroxides and tyrosyl radicals. The Mims ENDOR experiment is used at 94 and 263 GHz to detect small, isotropic hyperfine couplings in a range of 0.5 − 0.7 MHz at three trapped tyrosyl intermediates in "active" complexes of E. coli ribonucleotide reductase. The sharp spectral features give the first direct experimental evidence of hydrogen-bound water molecules at the radical intermediates, which are part of a long range proton-coupled electron transfer chain across different subunits of the enzyme. Small theoretical models are used to link the observed hyperfine couplings to a well defined, in-plane coordination of the water molecules. Very small amounts of spin density (∼ 0.01 %) on the oxygen nucleus, facilitated by the hydrogen-bond, are enough to cause detectable hyperfine splitting in the spectra. The exquisite capability of very high-field ENDOR spectroscopy to produce narrow spectral lines is shown and rationalized, allowing a reevaluation of our previous models of the tyrosyl radical intermediate Y•356. The thesis then answers the previously open question: Which hyperfine spectroscopy experiment is best suited to study 17O water around organic radicals ? The performance of three different types of hyperfine spectroscopy, recorded at 34 and 94 GHz EPR frequency, is compared for two nitroxide radicals as well as one tyrosyl radical. While all techniques detect 17O signals, the HYSCORE experiments at 34 GHz best show the presence of large hyperfine couplings in the range of 1 − 8 MHz for the two nitroxide radicals. Mims ENDOR experiments at 94 GHz best reveal small, isotropic couplings of 0.6 − 0.8 MHz for the nitroxide radical with a five-membered ring as well as the tyrosyl radical. Theoretical models and molecular dynamics simulations are used to show that large, anisotropic 17O couplings correspond to out-of-plane coordination while small isotropic couplings indicate in-plane coordination for all three radicals. All experiments performed in this thesis show that 17O hyperfine spectroscopy is a well suited method to detect water molecules at organic radicals. The strong dependence of 17O hyperfine coupling parameters on the hydrogen-bond geometry results in easily recognizable coupling structures, i.e. fngerprint signatures, of in-plane water binding.
Keywords: EPR; Hyperfine Spectroscopy; Water-binding; Ribonucleotide Reductase; Nitroxide Radicals; Tyrosyl Radicals; 17O