The Crystal Structure of the R52Q Mutant Demonstrates a Role for R52 in Chromophore pK_a Regulation in Photoactive Yellow Protein^†,‡

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK_a of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK_a regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK_a of the chromophore. R52 is found to flip out during the formation of PYP_M. The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK_a regulation during the photocycle.