Abstract
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 approximately 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: approximately 4.15 A, Mn(II)Fe(II); approximately 3.25 A, Mn(III)Fe(II); approximately 2.90 A, Mn(III)Fe(III); and approximately 2.75 A, Mn(IV)Fe(III). Shortening of the Mn/Fe-ligand bond lengths indicated formation of additional metal bridges, i.e. microO(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 approximately 2.90 A and approximately 2.75 A Mn-Fe distances, typical for di-microO(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.
Highlights
Ribonucleotide reductases (RNRs)3 are the only enzymes that, in all organisms, catalyze the reduction of ribonucleotides to their deoxy forms essential for DNA synthesis [1,2,3]
Metal Content and Site Occupancy—Two types of Chlamydia trachomatis (Ct) RNR protein samples were used in this spectroscopic study: (a) asisolated, oxidized R2 protein, denoted R2ox, and (b) the same R2 protein extensively reduced with DTT in an R1R2 complex, R2red
The metal contents of samples were quantified by total-reflection x-ray fluorescence detection (TXRF) and by previously described spectroscopy methods [32]; briefly, the manganese content was determined by EPR in an acid-denatured protein sample, and the iron content was spectrophotometrically determined using an iron complex assay)
Summary
Ribonucleotide reductases (RNRs) are the only enzymes that, in all organisms, catalyze the reduction of ribonucleotides to their deoxy forms essential for DNA synthesis [1,2,3]. Extensive investigations on Fe-Fe RNRs from, e.g. Escherichia coli, Saccharomyces cerevisiae, Mus musculus, and Homo sapiens have established that the catalytic reactions involve activation of an O2 molecule at the di-metal cluster to generate a high potential site, which oxidizes a nearby tyrosine residue to a tyrosyl radical, Y1⁄7 (8 –10). Subsequent proton-coupled electron transfer [13] leads to the re-reduction of Y1⁄7 and to the oxidation of a cysteine at the substrate binding site in R1 to a radical (C1⁄7) [14, 15]. Reactivation of the enzyme first requires reduction of the metal site to Fe(II), which must react with O2, leading to the cleavage of the O–O bond and again to the formation of Fe(III) and Y1⁄7 [22, 23]
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