| dc.contributor.advisor | Bennati, Marina Prof. Dr. | |
| dc.contributor.author | Nick, Thomas Udo | |
| dc.date.accessioned | 2016-06-16T07:48:56Z | |
| dc.date.available | 2016-06-16T07:48:56Z | |
| dc.date.issued | 2016-06-16 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0028-877F-7 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-5691 | |
| dc.language.iso | eng | de |
| dc.publisher | Niedersächsische Staats- und Universitätsbibliothek Göttingen | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 540 | de |
| dc.title | Hydrogen Bonds and Electrostatic Environment of Radical Intermediates in Ribonucleotide Reductase Ia | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Bennati, Marina Prof. Dr. | |
| dc.date.examination | 2015-06-29 | |
| dc.description.abstracteng | Ribonucleotide reductase connects the RNA and the DNA world via strictly controlled radical chemistry that reduces all four essential ribonucleotides to deoxyribonucleotides. In RNR Ia, the starting point is the µ-oxo diiron cofactor, where a “stable” tyrosine radical (Y122•) is formed from a nearby tyrosine in the β subunit. Successive studies showed that Y356•(β) Y731•(α) and Y730•(α) are intermediate steps of an intersubunit radical pathway, before a putative catalytic cysteine radical (C439•) is formed in the α subunit. Conformational gating hinders the direct observation of these transient radicals. A well-characterized mutation strategy by site-specific incorporation of the unnatural 3-amino-tyrosine (NH2Y) was successfully used to omit conformational gating. To analyze electrostatic effects and hydrogen (H) bond networks, all three Ys (Y356, Y731 & Y730) were successively mutated. Seminal studies revealed an exceptional difference between the tyrosine radials formed within the radical propagation and the Y122• at its beginning. The stepwise oxidation and reduction of these amino acid radicals is directly linked to a proton-coupled electron transfer (PCET). Therefore, the investigation of electrostatics and H bonds is fundamental to understand this important process in biology.
Pulsed 263-GHz EPR spectroscopy as well as ENDOR spectroscopy delivered insight based on closely characterized mutation approaches into the electronic and H bond structure of the NH2Ys•. It could be shown that an electropositive surrounding of moderate to strong H bonds are a common feature in α and β subunits. In the α subunit, double mutant approaches delivered insight into the effect of the removal of an H bond donor on the radical transfer efficiency and supported the assignment of the ENDOR studies. Deuteron nuclei (2H) ENDOR spectroscopy revealed 2, 1 and 0 H bonds perpendicular to the ring plane of NH2Y730•, NH2Y731• and NH2Y356•, which is consistent with a “π-stacking” between Y731 and Y730. Three structural DFT models for NH2Y731• based on optimized crystal structures have been discussed in terms of H bonds and environment. A perpendicular strong H bond (1.6 Å) and a weak H bond (≥1.9 Å) was consistent with the electrostatics observed at NH2Y731•. NH2Y356• showed the lowest gx value, typical for a polar electrostatic environment. Due to the limited structural data, no active model of NH2Y356• could be obtained. The possible influences on the gx value were discussed based on small model DFT calculations. Experimentally, one weak to moderate H bond (1.9±0.1 Å) could be resolved in the forward radical transfer to a wild type-β Y356• environment using a different mutation strategy. DFT models consistent with the obtained g values proposed another weak H bond (>2.1 Å). All moderate H bonds found at residue β-356 were in-plane of the tyrosine π system. Overall, this illustrates that different H bond networks in the α and β subunit are used to promote this long proton-coupled redox chain. | de |
| dc.contributor.coReferee | Groot, Bert de Prof. Dr. | |
| dc.contributor.thirdReferee | Lange, Adam Dr. | |
| dc.subject.eng | ribonucleotide reductase | de |
| dc.subject.eng | DNA-Synthesis | de |
| dc.subject.eng | PCET | de |
| dc.subject.eng | pi-stacking | de |
| dc.subject.eng | electron transfer mechanism | de |
| dc.subject.eng | high-field EPR | de |
| dc.subject.eng | high-field ENDOR | de |
| dc.subject.eng | multifrequency | de |
| dc.subject.eng | hydrogen-bonding | de |
| dc.subject.eng | 3-aminotyrosyl radical | de |
| dc.subject.eng | unnatural amino acid radicals | de |
| dc.subject.eng | protein interface | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0028-877F-7-6 | |
| dc.affiliation.institute | Fakultät für Chemie | de |
| dc.subject.gokfull | Chemie (PPN62138352X) | de |
| dc.identifier.ppn | 86153316X | |