Weitreichender Elektronentransfer in biologischen Systemen

Abstract

Ribonucleotide Reductases (RNR) are enzymes, which catalyze the reduction of all ribonucleotides to their deoxyribonucleotides and therefore play an important role in DNA repair und replication mechanisms. The class I RNR contains a stable diferric-tyrosyl radical cofactor which is essential for the reduction of the substrates. This radical can directly be observed by UV/Vis spectroscopy at 410 nm. After a substrate has bound in the active site, the radical will be transferred along a conserved hydrogen bridge across a distance of 35 A The RNR’s proton coupled electron transfer (PCET) is in the focus of this thesis. The goal was to gain insight in this process by stopped flow measurements and by laser flash photolysis of caged substrates. We could show that upon mixing cytidine diphosphate (CDP) and RNR no change of the tyrosyl radical signal can be observed in millisecond scale. To examine faster processes in the micro- and nanosecond scale, we synthesized two different caged substrates. We demonstrated that upon irradiation, photocleavage takes place and CDP is released, which can be converted to deoxycytidine diphosphate by RNR. After laser flash photolysis we observed no change of the tyrosyl signal at λ = 410 nm. This might be due the fact that the PCET is even faster than ~ 100 ns. Such a fast process is only in accordance with a hopping mechanism. The goal of a second project was to build up a system to measure the speed of an electron transfer in a double strand DNA-oligomer. We synthezised a modification based on thymidin, which will be reduced to its radical anion upon laser irradiation at λ = 308 nm. An incorporated porphyrin system acts as the electron acceptor and will be reduced to its radical anion. By time dependent observation of the porphyrins absorption, the kET of this electron hopping process can be measured. We could synthesize both modifications (T* and porphyrin) and they were incorporated into a double strand DNA-oligomer. We could show that upon irradiation of the porphyrin the chromophor is stable against photochemical processes and no phosphorescence takes place. Upon laser flash photolysis we observed a fast increase of the absorption at 445 nm followed by a slow decrease. Fitting of the measured curves showed that the ET is faster than kET ≥ 106 s-1 if only one A:T pair is between donor and acceptor. Future experiments with a larger distance between thymidine and porphyrin will yield in more precise results concerning the speed of the ET.