Findings in the Neutron Crystal Structure Analysis of a Bilin Reductase PcyA Complexed with its Substrate Biliverdin

Abstract

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the conversion of biliverdin (BV) to phycocyanobilin, a light harvesting pigment. This reaction consists of two sequential steps using two electrons and two protons in each step. Knowing the protonation states of the substrate BV and its surrounding residues is important to understand this unique reaction mechanism. To visualize the hydrogens (and protons) in this unique enzyme, we conducted neutron crystallography of the PcyA-BV complex. In the enzyme, BV bound to PcyA was in equilibrium between the neutral (BV) and the protonated (BVH⁺) forms. Both of the structures including the hydrogen atoms could be determined. The protonation of the nearby essential residue Asp105 was complemented with the BV protonation states. His88, an essential residue, was protonated to form a hydrogen bond to a lactam oxygen of the BV A-ring. His88 is also hydrogen-bonded to His74 via a hydronium ion. In this article, we also describe the story to grow the large crystals of the PcyA-BV complex and its neutron diffraction experiment.