Fig. 5.

Protein–chromophore interactions and bilin lactim tautomers in 2551g3. At physiological pHs, PCB in solution is neutral in the all-Z,syn configuration with two ionized propionate side chains. Upon binding to the apoprotein, both propionates form ion pairs with the cationic side chains of R930, K956, and H969 in the chromophore pocket. The A ring tilts toward the β-face as the bound chromophore engages multiple interactions with the β2–β3 loop. These include a strong H bond between the A ring carbonyl and the backbone amide NH of K915 along with protonation of the pyrrole ring system by E914. As a result, the anionic side chain of E914 forms a strong ion pair with the positively charged bilin system (Lactam All Nitrogens Protonated, Far Left structure). Formation of the thioether linkage via C943 may occur concomitantly or soon thereafter. Tautomerization to form Lactim 1 can occur via transient NH deprotonation, e.g., B ring NH, and proton transfer to the A ring carbonyl via the axial E914 carboxylate side chain. We envisage this A ring lactim is stabilized by H bonds with two nearby oxygen atoms, i.e., the backbone amide carbonyl of K915 and the D ring carbonyl, affording two Lactim 1 A ring deprotonated isomers. Additional tautomerization to form Lactim 2 may occur via a similar mechanism, via direct proton transfer from the B ring to the A ring pyrrole nitrogens or directly from the original Lactam. Lactim 3 can be generated by proton transfer via the A ring lactim to the D ring carbonyl. Although the positive charge can be readily delocalized on all four pyrroles, we favor an enrichment of far-red–absorbing Lactim 2 in FR/X CBCRs due to the A ring’s proximity to the negatively charge β-face.