Biophysikalische Untersuchungen des gekoppelten Elektronen/Protronen-Transfers in der rekombinanten Ribonukleotid-Reduktphase der Maus

• Antragsteller: Dr. Peter Paul Schmidt
• Fachliche Zuordnung: Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
• Förderung: Förderung von 2002 bis 2006
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5390454

Ribonucleotide reductases (RNR) are the only source for deoxyribonucleotides and thus DNA. Class I RNRs perform two interesting reactions: (i) oxygen activation by a diiron center (reconstitution) forming a stable tyrosyl radical, which is essential for (ii) the enzymatic reaction. Their kinetics may be followed by rapid freeze quench electron paramagnetic resonance (EPR) and optical stopped-flow technique. (i) The reconstitution kinetics of mouse and E. coli RNR are different, allowing the investigation of different steps on the reaction pathway and the accumulation of new intermediate(s) in mouse RNR. Preliminary results show a considerable H/D kinetic isotope effect in mouse RNR suggesting accumulation of a high-valent iron intermediate. Detailed kinetic measurements will reveal whether this proton transfer is coupled to an electron transfer. Further, site directed mutants on the putative hydrogen delivery pathway in mouse RNR will be investigated. (ii) The enzymatic reaction mechanism is radical-based and might comprise a coupled proton/electron transfer over 35 Å. We will try to trap novel radical intermediates on this transfer pathway and in the active site by single turnover measurements in H2O and D2O. Both reactions, (i) and (ii), will be slowed down by use of cryoprotectants and measurements at sub-zero temperatures (-30°C). The main goal is to trap catalytically competent radical intermediates during (i) and (ii) and to characterize them by multi-frequency EPR, ENDOR, ESEEM, and other suitable spectroscopic methods (Mössbauer, resonance Raman, etc.).

• DFG-Verfahren: Schwerpunktprogramme
• Teilprojekt zu: SPP 1071: Radikale in der enzymatischen Katalyse