QM/MM Investigation for Protonation States in a Bilin Reductase PcyA-Biliverdin IXα Complex

Herein we report quantum mechanical/molecular mechanical (QM/MM) studies to investigate the most probable protonation states of active site amino acids and bound substrate based on a recently reported neutron diffraction structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA) by Unno et al. This structure was considered to be bound in its initial state of biliverdin IXα (BV), which has the C-pyrrole ring in the deprotonated state. The protonation state of BV suggested by neutron and spectroscopic studies is a stable, two-electron reduced complex with a bound hydronium ion. Several ambiguities in the neutron structure were observed which prompted a further theoretical analysis of the structure. This structural investigation provides new understanding of the PcyA and BV protonation states not previously reported in the literature. Our calculations suggest that the hydronium ion (H₃ O⁺ ) is energetically unfavorable, preferentially protonating the neighboring His88 residue and that the C-ring of BV is not protonated.