Redox Intermediates of the Mn-Fe Site in Subunit R2 of Chlamydia trachomatis Ribonucleotide Reductase AN X-RAY ABSORPTION AND EPR STUDY

The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O-2 activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2(red)) showed an isotropic six-line EPR signal at g similar to 2 of the Mn(II)Fe(II) state. In oxidized R2 (R2(ox)), the Mn(III)Fe(III) state exhibited EPR g values of 2.013, 2.009, and 2.015. By XAS, Mn-Fe distances and oxidation states of intermediates were determined and assigned as follows: similar to 4.15 angstrom, Mn(II)Fe(II); similar to 3.25 angstrom, Mn(III)Fe(II); similar to 2.90 angstrom, Mn(III)Fe(III); and similar to 2.75 angstrom, Mn(IV)Fe(III). Shortening of the Mn/Fe-ligand bond lengths indicated formation of additional metal bridges, i.e. mu O(H) and/or peroxidic species, upon O-2 activation at the site. The structural parameters suggest overall configurations of the Mn-Fe site similar to those of homo-metallic sites in other R2 proteins. However, the similar to 2.90 angstrom and similar to 2.75 angstrom Mn-Fe distances, typical for di-mu O(H) metal bridging, are shorter than inter-metal distances in any R2 crystal structure. In diffraction data collection, such bridges may be lost due to rapid x-ray photoreduction of high-valent metal ions, as demonstrated here for Fe(III) by XAS.