Abstract
Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms. The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical. Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions. In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron. The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1. There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies. Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study. Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible. Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2.
Highlights
The enzyme ribonucleotide reductase (RNR)1 catalyzes the reduction of the four ribonucleotides to the corresponding de
All of these proteins have dinuclear iron centers that bind dioxygen, and together they form a family called diiron-oxygen proteins [14], but their biological roles are very different (11, 14 –17); MMOH oxidizes methane to methanol in methanotrophic bacteria, ⌬9-D introduces a double bond into saturated fatty acids, and the R2 subunit of RNR generates, as mentioned above, a tyrosyl radical essential for the reduction of ribonucleotides to deoxyribonucleotides in eukaryotes and many bacteria and viruses
R2 from both mouse and Escherichia coli belongs to the same class of RNR, the sequence identity is only ϳ20% [18], and the iron-oxygen cluster in mouse R2 is much more labile compared with E. coli R2 [19]
Summary
All commercial reagents were used as obtained: sodium acetate (Sigma), ammonium iron(II) sulfate ((NH4)2Fe(II)(SO4)21⁄76H2O) (Merck), methylviologen (1,1Ј-dimethyl-4,4Ј-bipyridinium dichloride hydrate) (Sigma), sodium dithionite (Aldrich), 96% glycerol (Prolabo), phenosafranine (Aldrich), sodium L-ascorbate (Sigma), and dipotassium hydrogen phosphate/potassium dihydrogen phosphate (Merck). Cloned mouse apo-R2 protein was prepared and purified as reported earlier [37]. The protein was crystallized using the hanging drop vapor diffusion method at pH 4.7 according to the previously published protocol [38]. The crystals were shaped like thin sheets with a size of up to 0.05 ϫ 0.10 ϫ 0.50 mm and belong to space group C2221
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