Abstract
Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.
Highlights
Ribonucleotide reductase (RNR) is an essential enzyme in all domains of life
We report several crystal structures of B. anthracis NrdF—the radical-generating subunit of class Ib RNR
As for the other class Ib NrdF proteins characterized to date, Bacillus anthracis (Ba) NrdF is extremely sensitive to X-ray photo-reduction
Summary
Ribonucleotide reductase (RNR) is an essential enzyme in all domains of life. It catalyzes ribonucleotide reduction to the corresponding deoxyribonucleotides, which are further used in DNA synthesis and repair [1]. RNRs are complex enzymes that utilize radical chemistry for their catalytic activity. RNRs are grouped into classes I, II, and III (reviewed in [2, 3]). Class I RNRs are oxygen-dependent and employ a radical-generating subunit denoted β or R2. The radical is transferred in excess of
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