Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases

Abstract

Class I ribonucleotide reductases consisting of R1 and R2 subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by reaction involving di-manganese ions in the R2 subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the R2 subunits with no external support from another subunit. Despite much work on the R2 subunits of Class Ib ribonucleotide reductases, also referred as NrdF, and its partner NrdI, the structural details of free radical generation remain largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidized and reduced forms, and similarly those of NrdF2: NrdI complex (NrdF2I). These structures provide the first atomic view of the mechanism of free radical generation in the R2 subunit. We propose that oxygen molecule accesses FMN through a well-formed channel in NrdI, seen clearly in the crystal structure, and upon electron transfer is converted to a superoxide ion. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. A delocalised Mn ion in the R2 subunit is seen in the electron density, which attacks Tyr 110 to produce a Tyr* free radical. Finally, a solvent channel to the dimanganese-binding site is observed to complete the cycle. The study therefore provides important structural clues on the initiation of free radical generation in the R2 subunit of the ribonucleotide reductase complex. Significance statementRibonucleotide reductases generate the deoxyribonucleotide pool in the cell for DNA replication and repair. The enzymes utilise free radical mechanism, where the mechanism of radical formation defines different classes of ribonucleotide reductases. Class Ib ribonucleotide reductases generate the free radical though di-manganese chemistry, assisted externally by NrdI. We describe here structural features required to achieve this mechanism. The structures clearly show a tunnel for oxygen access to the FMN site, tunnel to transport the consequent superoxide radical that is formed, a dislocated activated Mn, which appears to coordinate with a Tyrosine residue to form Tyr* radical and a water channel to complete the reaction cycle, thus enhancing our understanding of the steps of free radical generation.