Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å x-ray data

Abstract

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122 • ). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-Å x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122 • obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography. R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides and are thus essential for DNA synthesis. Class I RNRs consist of two homodimeric proteins: R1, which contains the active site and binding site for allosteric regulators, and R2, which generates and harbors the tyrosyl radical Y122• (Escherichia coli numbering) needed for catalysis. The catalytic reaction in R1 is believed to be initiated by a reversible radical transfer from Tyr 122 in R2 to an active-site cysteine in R1.The tyrosyl radical on the R2 subunit is generated by means of the reductive cleavage of molecular oxygen at a diiron center. Crystal structures of E. coli R2 are available for both the reduced diferrous (Fe 2). The structural data have, together with kinetic data and theoretical calculations, served as the basis for the formulation of proposals for the mechanism of radical generation and radical migration in the overall RNR reaction (3-9). However, no structure has been available for the radical-containing form, hence, the orientation and location of the active radical Y122• have not been known. The diiron center is in the active enzyme in the diferric form and couples antiferromagnetically to an S ϭ 0 ground state (3-5, 10). Detailed high-field EPR experiments have been performed on Y122• in frozen R2 solutions (11-15). The obtained g-tensor values were found to be indicators for the polarity of the radical environment. In particular, it was found that the tyrosyl radical is hydrogen-bonded in RNR of mouse and herpes simplex virus (12, 14), whereas in E. coli it is not (11-15).In...

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

“…This may be taken as evidence that terminal and/or metal-bridging O(H) ligands in CtR2 are integrated in hydrogen-bonding interactions, for example, with water molecules. Water molecules in hydrogen-bonding distance, for instance, to metal-binding carboxylates, indeed were found in high-resolution R2 structures (39,99,100). The proton distribution in XPR-induced structures thus may significantly differ from the respective native oxidation states.…”

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

“…1). All ligands of the iron center are well defined by the electron density, and their conformations are comparable to the structure of the wild-type oxidized diferric R2 (metR2) (42,43). There are, however, some noticeable differences.…”

“…The differences between these two water ligands are even more pronounced in subunit A (B-factor 33 versus 14 Å 2 , coordinating distance 2.7 versus 2.2 Å) indicating lower occupancy for Wat3 in subunit A. The high resolution structure of wild-type metR2 indicates 5-coordinated Fe1 (43). The presence of the extra water ligand to Fe1 may be made possible partly by the shift of Asp 84 toward His 122 described above.…”

“…(43). In wild-type metR2 the carboxyl group of Asp 84 is a monodentate ligand to Fe1 with the free carboxylate oxygen making a H-bond to the water coordinated to Fe2, the Fe1-bound oxygen making a second weak H-bond to Y122-OH (43). In R2-Y122H, D84 is rotated further away from the iron site, leaving one oxygen atom ligated to Fe1 and the free oxygen forming a new short hydrogen bond to N⑀ of His 122 (2.7-Å O-N distance).…”

Crystallization and preliminary X-ray analysis of the small subunit (R2F) of native ribonucleotide reductase fromCorynebacterium ammoniagenes

“…To date, several crystal structures of E. coli and mouse R2 or Mycobacterium tuberculosis and C. ammoniagenes R2F expressed in E. coli have been studied in various oxidation states and forms obtained by site-directed mutagenesis or by metal substitution (Nordlund et al, 1990;Logan et al, 1996;Voegtli et al, 2000;Hö gbom et al, 2002Hö gbom et al, , 2003Uppsten et al, 2004;Strand et al, 2004;Lendzian, 2005). Recently, the structure of the Mn/Fe metallo-cofactor of the chlamydial RNR has been studied by extended X-ray absorption finestructure spectroscopy (Younker et al, 2008).…”