Abstract A recently discovered subgroup of class I ribonucleotide reductase (RNR) found in the infectious bacterium Chlamydia trachomatis ( C. trachomatis) was shown to exhibit a high-valent Fe IIIFe IV center instead of the tyrosyl radical observed normally in all class I RNRs. The X-ray structure showed that C. trachomatis WT RNR has a phenylalanine at the position of the active tyrosine in Escherichia coli RNR. In this paper the X-ray structure of variant F127Y is presented, where the tyrosine is restored. Using 1H- and 57Fe-ENDOR spectroscopy it is shown, that in WT and variants F127Y and Y129F of C. trachomatis RNR, the Fe IIIFe IV center is virtually identical with the short-lived intermediate X observed during the iron oxygen reconstitution reaction in class I RNR from E. coli. The experimental data are consistent with a recent theoretical model for X, proposing two bridging oxo ligands and one terminal water ligand. A surprising extension of the lifetime of the Fe IIIFe IV state in C. trachomatis from a few seconds to several hours at room temperature was observed under catalytic conditions in the presence of substrate. These findings suggest a possible new role for the Fe IIIFe IV state also in other class I RNR, during the catalytic radical transfer reaction, by which the substrate turnover is started.