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Research has been conducted on the ribonucleotide reductase which catalyzes deoxyribonucleotide biosynthesis. Results suggest that sulfinyl radicals are important intermediates in the ribonucleotide reductase radical chemistry.

A study was conducted to analyze the interconversion and metabolic fate of 8-oxoguanine-containing ribonucleotides in mammalian cells. Bio-Rad protein assay kits were utilized with bovine serum albumin to obtain protein concentration. Experimental results indicated that the ribonucleotide reductase may prevent the influx of potent genotoxic substrates from the oxidized ribonucleotide pool.

Researchers have investigated the mixed-valent states of the recombinant mammalian (mouse) and HSV1 R2 proteins. An EPR study looked at the diiron sites in mouse and HSV1 R2 proteins obtained by reduction in frozen solution at 77 K with mobile electrons produced by gamma-radiolysis. It was established that gamma-irradiation of mouse and HSV1 R2 proteins in a water-glycerol glass at 77 K leads to transient mixed-valent diiron intermediates in quite high yields.

The binding of the nucleoside triphosphates ATP, dATP, dTTP and dGTP effectors helps in the formation of the R1 homodimer protein which is necessary for binding to the R2 homodimer to form the mouse ribonucleotide reductase. The binding of ATP or dATP to the active site of the enzyme regulates enzyme activity. The regulation occurs through control of electron transport between the catalytic site of the subunit R1 and the tyrosyl free radical of the subunit R2, rather than a change in the aggregation state of R1/R2.

Nonidentical protein subunits, R1 and R2, are each inactive in the absence of the other, and constitute the mouse ribonucleotide reductase. Active enzyme complexes are formed by the carboxyl-terminal region of the R2 protein. The R2 protein also produces sharp H1 nuclear magnetic resonance spectroscopic lines. The lines are much narrower than the resonance lines. Reduction in the broadening of the spectral lines indicates molecular segmental mobility.

Comparative studies of the metal content and specific activity of the Chlamydia trachomatis (C. tm.) wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli are reported. The results indicated that the affinity of the RNR R2 proteins for binding metals is influenced by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins.

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an [[Alpha].sub.2][[Beta].sub.2] form of RNR consisting of two homodimeric subunits, proteins R1 ([[Alpha].sub.2]) and R2 ([[Beta].sub.2]). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p ([Beta]') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a [Beta][Beta]' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an [[Alpha].sub.2][Beta][Beta]' heterotetramer, where Rnr4p plays an important role in the interaction between the [[Alpha].sub.2] and the [Beta][Beta]' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

Diiron clusters in the R2 proteins of mouse and herpes simplex virus 1 ribonucleotide reductase (HSV1 RNR) undergo mild reduction to produce two mixed-valent (Fe(II)Fe(III)) clusters. EPR spectroscopy proves the high yield of this mixed-valent state. The redox properties are different from those of R2 proteins from Escherichia coli. The E. coli iron center is less reactive and more strongly-attached to the R2 protein than in HSV1/2 and mouse.