Figure 1.

The proposed radical initiation pathway and its energetics in E. coli RNR with site-specific incorporation of unnatural amino acids (13). Residues in grey are associated with the diferric cluster, in black are proposed pathway residues (13), and in blue are the unnatural amino acids (F₃Y and NH₂Y) to probe the pathway in the present studies. Note that the structural location of Y₃₅₆ is unknown. (C) Peak potentials (E_p) for free (NH₂Y), and N-acetylated and C-amidated (Y, W, F₃Y) amino acids, as a function of pH. The E_ps for Y•, W• and 2,3,5-F₃Y• have been previously determined (25) and the trace for NH₂Y• has been generated from the reduction potential determined at pH 7, assuming Nernstian behavior (27). (D) The E_ps from panel (C) have been assigned to residues in the radical propagation pathway to provide a qualitative energy landscape. Red and blue rectangles represent the peak potentials for each amino acid at pH 7.0 and 8.4, respectively. The peak potentials of WH•+ and F₃Y• are represented by red rectangles with blue diagonal lines as they do not change between pH 7–8.4. The E_p range for Y₁₂₂• is expanded (indicated by shading), because its properties relative to the other three Ys, including its pK_a, are unique. Y₁₂₂• has a half-life of ∼4 days and likely represents a thermodynamic hole (45). Brackets are placed around W₄₈ as no direct evidence is available that places it on the pathway. If it is on the pathway, its protonation state, W₄₈H•+ vs. W₄₈•, that participates in radical transfer is unknown, and therefore E_ps for both of these species are included.