Mechanism of Radical Initiation and Transfer in Class Id Ribonucleotide Reductase Based on Density Functional Theory.

Class Id ribonucleotide reductase (RNR) is a newly discovered enzyme, which employs the dimanganese cofactor in the superoxidized state (Mn/Mn) as the radical initiator. The dimanganese cofactor of class Id RNR in the reduced state (inactive) is clearly based on the crystal structure of the -β subunit. However, the state of the dimanganese cofactor of class Id RNR in the oxidized state (active) is not known. The X-band EPR spectra have shown that the activated -β subunit exists in two distinct complexes, and . In this work, quantum mechanical/molecular mechanical calculations were carried out to study class Id RNR. First, we have determined that complex contains a Mn-(μ-oxo)-Mn cluster, and complex contains a Mn-(μ-hydroxo/μ-oxo)-Mn cluster. Then, based on the determined dimanganese cofactors, the mechanism of radical initiation and transfer in class Id RNR is revealed. The Mn-(μ-oxo)-Mn cluster in complex has not enough reduction potential to initiate radical transfer directly. Instead, it needs to be monoprotonated into Mn-(μ-hydroxo/μ-oxo)-Mn (complex ) before the radical transfer. The protonation state of μ-oxo can be regulated by changing the protein microenvironment, which is induced by the protein aggregation and separation of β subunits with α subunits. The radical transfer between the cluster of Mn-(μ-hydroxo/μ-oxo)-Mn and Trp30 in the radical-transfer chain of the -β subunit (Mn/Mn ↔ His100 ↔ Asp194 ↔ Trp30 ↔ Arg99) is a water-mediated tri-proton-coupled electron transfer, which transfers proton from the ε-amino group of Lys71 to the carboxyl group of Glu97 via the water molecule Wat551 and the bridging μ-hydroxo ligand through a three-step reaction. This newly discovered proton-coupled electron-transfer mechanism in class Id RNR is different from those reported in the known Ia-Ic RNRs. The ε-amino group of Lys71, which serves as a proton donor, plays an important role in the radical transfer.