Photo-isomerization upshifts the pKa of the photoactive yellow protein chromophore to contribute to photocycle propagation
The influence of chromophore structure on the ionization constant of Photoactive Yellow Protein chromophore is explored with isolated para-coumaric acid (pCA) and thiomethyl-para-coumaric acid model chromophores in solution. pH titration coupled with visible absorption spectra of the trans and photogenerated cis forms of pCA demonstrated that isomerization of the chromophore in solution increases the pKa of the phenolate group by 0.6 units (to 10.1+/-0.23). Formation of the pCA thioester reduces the pKa of the phenolic group by 0.3 units (from 9.5+/-0.18 to 9.2+/-0.15), but a macroscopic cis-TMpCA population was not achieved. These trends were reproduced with electronic structure calculations, which demonstrated by a Natural Bond Orbital analysis that the pKa upshift for the cis configuration vs. the trans configuration is attributed to increased Columbic repulsion. This structurally induced pKa upshift after isomerization facilitates the subsequent protonation of the chromophore within PYP and propagates the photocycle response and consequently the in vivo photo-activity.