Absorption spectra and reaction changes of two mutants of the bilin reductase PcyA and their correlation with the protonation states

PcyA is an enzyme that biosynthesizes phycocyanobilin, a pigment used in photosynthesis and photosensing from the heme metabolite biliverdin. A single residue mutation of key amino acids within PcyA alters the electronic absorption spectra in the state in complex with biliverdin. The differences in the spectra have been proposed to reflect differences in the protonation states of the biliverdin bound to the PcyA mutants, however, to date, there have been no examples of the protonation states of the biliverdin bound to the PcyA mutants being visualized. In this study, the protonation states near the biliverdin-binding site of the two mutants was successfully visualized and proved the hypothesis by a combination of neutron crystallography and computational chemistry. (author)