Class Id ribonucleotide reductase utilizes a Mn₂(IV,III) cofactor and undergoes large conformational changes on metal...

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Structural Biology

Background:

• High-valent manganese cofactors are uncommon in biological systems, with photosynthetic machinery being a notable exception.
• Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis, and a recently identified subclass, class Id, is proposed to utilize a Mn2(IV,III) cofactor.

Purpose of the Study:

• To investigate the enzymatic relevance and structural characteristics of the Mn2(IV,III) cofactor in class Id NrdB.
• To elucidate the mechanism of Mn2(IV,III) cofactor formation and activation in class Id NrdB.

Main Methods:

• Electron paramagnetic resonance (EPR) spectroscopy and enzyme activity assays were employed to study the Mn2(IV,III) cofactor in Facklamia ignava NrdB.
• X-ray crystallography was used to determine the structures of apo- and Mn-loaded Leeuwenhoekiella blandensis NrdB.
• In vitro reconstitution experiments were performed to investigate cofactor formation using different oxygen species.

Main Results:

• The presence and quantity of the Mn2(IV,III) cofactor directly correlate with the catalytic activity of class Id NrdB.
• X-ray structures revealed a dimanganese center and significant conformational changes in L. blandensis NrdB upon metal loading, including loop refolding and helix reorientation.
• The Mn2(IV,III) cofactor is generated from reduced oxygen species, specifically hydrogen peroxide and superoxide, with superoxide being a more efficient activator.

Conclusions:

• The Mn2(IV,III) cofactor is enzymatically crucial for class Id NrdB activity.
• Class Id NrdB undergoes substantial conformational changes upon manganese cofactor binding, facilitating substrate access.
• Superoxide is proposed as the likely physiological species involved in the activation of the high-valent manganese cofactor in class Id RNRs.