Enhanced Quantum Simulations Reveal Conformation-Controlled, Site-Resolved Proton Binding in Biliverdin

For a molecule with several titratable sites, a measured pKa reports a change in the number of bound protons, but not the population of each tautomer. The biological chromophore biliverdin (BV) is a longstanding example: the protonation of its four pyrrole nitrogens controls its photoactivity, yet the reported pKa values vary by about two units, and ring C is typically assumed deprotonated in neutral BV without a site-resolving measurement. BV is also flexible, so its protonation microstates and its conformers form coupled ensembles: the shape of the molecule changes how tightly it binds the proton. Here, we compute the macroscopic pKa of BV in explicit water and the populations of its four neutral tautomers, from the site-resolved pKa of the four rings. Enhanced quantum-mechanical simulations follow a proton into bulk water, and the free energies of proton transfer between rings and of ring rotation connect all protonation states in one thermodynamically consistent network. In the locked-helix conformation that dominates in water, BV has a macroscopic pKa of 4.0, and its four nitrogens are almost equally basic (site pKa values of 4.5 for rings B and C and 4.9 for rings A and D). No single tautomer dominates: the tautomers deprotonated at rings B and C each make up 36% of neutral BV, and those deprotonated at rings A and D 14% each. Opening the helix at ring D, as in bacteriophytochromes, raises the macroscopic pKa to 6.4 and shifts the neutral form almost entirely to the ring B (71%) and ring C (27%) tautomers. The conformation of BV thus controls its proton binding more than the choice of ring.