Open Access

Melanie M. Hertel

• In the first part of the present work (Chapter 3), EPR spectroscopy at different microwave frequencies, namely at 9 GHz (X-band), 34 GHz (Q-band) and 180 GHz (G-band), was employed to resolve the g-values and the HFCs of a putative radical intermediate involved in the reduction of benzoyl-CoA catalyzed by benzoyl-CoA reductase. In particular, the effect of 33S-labeling on the EPR line shape was studied at X- and Q-band frequencies in order to gain further evidence for a sulfur centered radical proposed to be the electron donor in the reduction or the aromatic ring of BCoA [I]. The spectral components observed at X-, Q- and G-band were overall consistent and showed at least three overlapping EPR signals. The signal postulated to be due to a disulfide radical anion showed no resolved g-values and a relaxation behaviour faster than expected for such a radical species. These observations together with the simulations suggest that the signal could arise from a radical exchange coupled to an [4Fe-4S] cluster located nearby. In the future, pulsed EPR and ENDOR spectroscopy on the 57Fe-labeled enzyme could help to solve this question. The potential of high-field ENDOR in combination with 13C- and 31P-labeling for investigating the structure at the active site in proteins could be verified in the studies of the ligation sphere of the cofactor Mn2+ in Ras as reported in Chapter 4 [2]. Therein, high-field ENDOR performed at 94 GHz (W-band) was used to detect the hyperfine interactions between the electron spin mainly located on the metal ion and the phosphorous nuclei of the bound GDP and GppNHp as well as the carbon nuclei of bound amino acids in the wild-type Ras protein and its oncogenic mutant G12V. These studies aimed at searching for an additional free phosphate ion or amino acid ligand bound to the metal center in the wild type GDP-bound protein with respect to its oncogenic mutant. Rom the 13C- and 31P-ENDOR spectra, the hyperfine couplings of directly bound amino acids and the bound nucleotides were compatible with the hyperfine couplings obtained from DFT calculations based on the crystal structure data. No differences in the 13C- and 31P-ENDOR spectra could be found for the wild-type GDP-bound protein in comparison to its oncogenic mutant in frozen solution. Therefore, no evidence for binding of an additional free phosphate ion or amino acid ligand in the wild-type GDP-bound protein was found. The distances between the detected nuclei and the meta1 ion were in agreement with the ones extracted from crystal structures reported in the literature. Future 35C1-ENDOR studies could clarify whether a chloride ion from the buffer solution could be the ligand replacing one water molecule in the wild type GDP-bound Ras. In Chapter 5, the implementation of a high-field ENDOR setup into a homebuilt pulsed EPR spectrometer operating at 180 GHz is reported and its performance for 1H-ENDOR demonstrated on the model system BDPA. Mims and Davies ENDOR spectra were also obtained for Ras(wt).Mn2+.DP. The increased nuclear Zeeman resolution at 180 GHz may be further exploited in the future by extending the setup for studying hyperfine couplings of low-y nuclei such as 33S, 15N , 17O or 2H. In the present work, the advantages of performing EPR and ENDOR experiments at high fields and frequencies could be nicely demonstrated with the 94 GHz ENDOR studies of Ras. Furthermore, the complementing information obtained at X- and Q-band frequencies in the multifrequency EPR studies on BCR demonstrated that the analysis of EPR spectra can be greatly facilitated by simulating the spectra measured at different MW frequencies with the same set of parameters consistent with a proposed radical. Overall, it could be shown that the use of different experimental techniques at multiple fields and frequencies renders EPR spectroscopy a powerfull tool for structural studies in biological systems.
• Die vorliegende Arbeit befasst sich mit der Identifizierung von radikalischen Intermediaten, welche an der Reduktion von Benzoyl-CoA (BCoA) durch Benzoyl- CoA-Reduktase (BCR) beteiligt sind, sowie mit strukturellen Untersuchungen der aktiven Bindungsstelle im G-Prcrtein Ras mit Hilfe von elektronenparamagnetiseher Resonanz- (EPR) und Elektronen-Kern-Doppelresonanz- (ENDOR) Spektroskopie. Die zahlreichen Vorteile, EPR- und ENDOR-Experimente bei hohen Frequenzen und Feldern durchzuführen, im besonderen an Hochspin-Systemen wie Mn(II), lieferten die Motivation, einen ENDOR-Aufbau in ein gepulstes Eigenbau-180-GHz-EPR-Spektrometer zu implementieren. EPR-spektroskopische Methoden haben sich in der Vergangenheit erfolgreich bewährt, um die Struktur und Funktion von aktiven Zentren in Metallproteinen zu untersuchen [3, 41. Mit Hilfe von EPR-Spektroskopie können Hyperfeinkopplungen zwischen dem Elektronenspin und Kernspins von Kernen in der Nähe des paramagnetischen Metallions gewonnen werden [5, 61. Hyperfeinkopplungen stellen eine bedeutende Informationsquelle bei der Untersuchung katalytischer Mechanismen, der Struktur sowie der Koordinationsgeometrie von paramagnetischen Metallzentren dar. Die Hyperfeinkopplungen können entweder aus der Aufspaltung der EPR-Linien oder über Doppelresonanzmethoden wie z.B. ENDORSpektroskopie gewonnen werden. In einem ENDOR-Experiment wird das kernmagnetische Resonanz- (NMR) Spektrum von Kernen, welche mit dem paramagnetischen Zentrum wechselwirken, über die Änderung der EPR-Signalintensität durch Anregung der Kernübergänge bei Radiofrequenz-Einstrahlung aufgenommen.