Fig. 4.

The NrdI-NrdF channel. (A) NrdI-NrdF complex formation extends the NrdF active-site channel to the FMN cofactor. The complex channel is depicted as a light blue mesh and was calculated using a 1.4 Å probe radius. Selected NrdI (green) and NrdF (white) residues lining the channel are shown as sticks. (B) Observation of a trapped species, best modeled as peroxide, in the NrdI-NrdF channel in a crystal reduced by dithionite in the presence of oxygen (NrdI_hq-NrdF_perox). Strong F_o − F_c electron density (green mesh, contoured at 3.3σ) is present in the channel after the first refinement cycle. The FMN cofactor (yellow), NrdI side chains lining the channel (purple), NrdF residues in the channel and at the active site (white), and the peroxide (red) are all shown as sticks. (C) A magnified view of the modeled peroxide shown in Fig. 3B and hydrogen bonding interactions with residues and solvent in the channel. The final 2F_o − F_c electron density (blue mesh, contoured at 1.8σ) is superimposed on the initial F_o − F_c electron density map from Fig. 3A. Water molecules are shown as red spheres. Dashed black lines indicate potential hydrogen bonding interactions. The gray dashed line represents the distance between the modeled peroxide and the nearest charged residue, conserved NrdF residue Lys²⁶⁰. The Glu¹⁹² backbone carbonyl group and the side chain of Ser¹⁵⁹ constitute the narrowest point of the active-site channel. The oxygen atom distal to the Mn^II₂ site interacts strongly with the side chains of NrdI residues Asn⁸⁵ (2.8 Å) and conserved Asn⁸³ (2.8 Å). (D) The extended hydrogen bonding network near the putative peroxide binding site. The side-chain orientations of Asn⁸³ and Asn²⁵⁷ can be assigned unequivocally based on their interactions with Lys²⁶⁰ and the backbone amide nitrogen of Asn⁸⁵, and the carbonyl oxygen of Phe²⁵³, respectively. The interactions of w2 with Asn²⁵⁷ (2.8 Å) and the backbone carbonyl of Ser¹⁵⁹ (2.7 Å) constrain w2 to act as a hydrogen bond acceptor for the proximal oxygen atom of the modeled peroxide (2.9 Å), suggesting that this oxygen is protonated. The distal oxygen accepts two hydrogen bonds from Asn⁸³ and Asn⁸⁵. Because no other potential hydrogen bond donor or acceptor exists for this oxygen atom, its protonation state cannot be determined from this analysis.