Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class la Binuclear Non-Heme Iron Enzyme

Abstract

The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe hetero-binuclear cofactor, rather than the Fe/Fe cofactor found in the beta (R2) subunit of the class Ia enzymes, to react with O-2. This reaction produces a stable (MnFeIII)-Fe-IV cofactor that initiates a radical, which transfers to the adjacent alpha (R1) subunit and reacts with the substrate. We have studied the (MnFeIII)-Fe-IV cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe-III, whereas MCD reflects the spin-allowed transitions mostly on the Mn-IV. We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn-IV at the site proximal to Phe(127). Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH- to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the (MnFeIII)-Fe-IV cofactor as having a mu-oxo, mu-hydroxo core and a terminal hydroxo ligand on the Mn-IV. From DFT calculations, the Mn-IV at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class La RNR, and the OH- terminal ligand on this Mn-IV provides a high proton affinity that could gate radical translocation to the alpha (R1) subunit.