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...

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.