We recently showed that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis uses a MnIV/FeIII cofactor to generate protein and substrate radicals in its catalytic mechanism [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the MnII/FeII cluster and O 2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second-order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active Mn IV/FeIII-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than MnIV/FeIII. Mössbauer spectra show that the intermediate contains a high-spin FeIV center. Its chemical and spectroscopic properties establish that the intermediate is a Mn IV/FeIV-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the MnIV (S Mn = 3/2) and high-spin FeIV (SFe = 2) sites.
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