Electron Paramagnetic Resonance and Nuclear Magnetic Resonance Studies of Class I Ribonucleotide Reductase

Abstract

Ribonucleotide reductase catalyses the reduction of ribonucleotides to the corresponding deoxyribonucleotides needed for DNA synthesis. This review describes recent studies on the iron/tyrosyl free radical site in the R2 protein of iron-containing (class I) ribonucleotide reductases. The active enzyme is composed of two homodimeric proteins, R1 and R2. Active protein R2 contains a diiron-oxygen site and a neighboring free radical on a tyrosyl residue per polypeptide chain. The properties of the different redox states of the diiron center in protein R2 are discussed, as well as the formation of the iron/radical site and its possible involvement in long range electron transfer from the substrate binding site in protein R1. The EPR properties of oxidized neutral tyrosyl free radicals are described, and also of tryptophan free radicals found in studies of a mutant of the R2 protein, which lacks the tyrosyl radical site. NMR studies on protein R2 include observations of paramagnetically shifted resonances. Structural NMR studies have been performed on its highly mobile C-terminal domain as well as the corresponding oligopeptide which interacts with protein R1.

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

“…2A) and 8 ms (9). In contrast to the wild type, the mutant radical spectrum shows a large doublet splitting similar to the tyrosine radical observed for Y122* of E. coli RNR (36).…”

“…Experimental parameters for all spectra: T ϭ 80 K, microwave power ϭ 5 W, modulation amplitude 0.2 mT, modulation frequency 10 kHz, microwave frequency 94.000 GHz, field calibrated with lithium/LiF to an accuracy of 5 ppm, each spectral trace obtained by numerical summation of 20 -22 single scans (80 s each) corresponding to a total accumulation time of 30 min. 47) and in aerobic RNR (36,48). They were also found to be transient intermediates in a variety of enzymes, such as peroxidases (31), cytochrome c oxidase (49), and in DNA photolyase, where, depending on the organism, tryptophan or tyrosine radicals have been observed (50).…”

Ribonucleotide Reductases

“…Several excellent reviews (31,32,37) have discussed the R2 structure, including that of its µ-oxo bridged dinuclear iron center. Briefly, the two µ-oxo-linked irons of the oxidized center are coordinated by four carboxylates and two histidines.…”

“…The generation of the tyrosyl radical is intimately linked to the center (reviewed in [30][31][32]. Either the iron-free apoR2 or metR2, the form that lacks the radical but has retained an intact diferric center, can be the starting point for radical formation.…”

“…Despite these differences we can distinguish a pattern that suggests a common evolutionary origin for the classes. Several excellent reviews of ribonucleotide reduction have been published during recent years, often stressing special aspects such as radical formation in class I and class II enzymes (30), metal centers (31,32), enzyme regulation (33), evolution (34), or mammalian reductases (35).…”