Biophysical studies of ribonucleotide reductase

Detta är en avhandling från Stockholm : Stockholm University

Sammanfattning: Ribonucleotide reductase (EC 1.17.4) catalyses the enzymatic reduction of ribonucleotides to their corresponding deoxyribo- nucleotides, the monomeric precursors for the DNA synthesis in all living cells. All ribonucleotide reductases so far Isolated from different species have been found to contain metal ions, either iron, cobolt or manganese. This thesis deals with iron containing ribonucleotide reductases, which can be isolated originating from certain bacteria, bacteriophages, mammalian cells and viruses.Iron containing ribonucleotide reductases consist of two non identical subunits, one of which carries a stable tyrosine free radical and a p-oxo bridged iron center. In this work ribonucleotide reductase from E. colt, bacteriophage T4 and mammalian cells has been investigated. The tyrosyl radical which is a prerequisite for enzymatic activity can be studied by EPR. Radicals from different species exhibit slightly different EPR signals. The difference is shown to emanate from minor changes in configurations of the tyrosyl residue harbouring the unpaired electron, reflecting differences in the amino acid sequence in enzymes from different species.NMR studies of the iron containg subunit of the E. colt enzyme show two paramagnetically shifted proton resonances. The protons giving rise to one of the resonances exchanges with water. This resonance is suggested to originate from histidine residues ligated to the iron center. The other resonance has not been assigned but most probably emanates from ß and nr methylene protons of aspartic and/or glutamic acids liganded to the iron center.The tyrosyl radical is close to the iron center but is probably not a direct ligand to the iron ions. The proximity is reflected by the EPR relaxation behaviour of the tyrosyl radical. An interaction between the iron center and the radical enhances the relaxation of the latter. The enhancement is different in mammalian and E. colt ribonucleotide reductase, again reflecting slight differences in the exact sterical arrangement of the tyrosyl radical-iron center. The E. colt radical is proposed to relax through a Raman process and by a mechanism based on dipolar interaction. The mammalian radical exhibits an even more enhanced relaxation behaviour than the E. colt radical and may also relax through an Orbach process made possible by a mix of the wave functions of the tyrosyl radical and the iron center.

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