Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: A pH-driven color transition

Photoactive yellow protein (PYP) is a blue light sensor present in the purp le photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) o f 446 nm. The anionic p-hydroxycinnamoyl chromophore of PYP is covalently b ound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydroge n-bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becom es protonated in the photobleached state (I-2) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and t he chromophore and partial exposure of the phenolic ring to the solvent. In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a ke y factor in controlling the color and photocycle kinetics of PYP is this H- bonding system. To further investigate this, we have now characterized Glu4 6Asp and Glu46Ala mutants. The ground-state absorption spectrum of the Glu4 6Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species : a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-sh ifted deprotonated (basic) form (lambda(max) = 444 nm). Both of these speci es are photoactive. A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive redshifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm). We attribute these spectral transitions to protonation/deprotonation o f the phenolate oxygen of the chromophore. This is demonstrated by FT Raman spectra. Dark recovery kinetics (return to the unphotolyzed state) were fo und to vary appreciably between these various photoactive species. These sp ectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.