Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein

TO understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP), Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic revel. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein, Glu46, replaced by Gin, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state, The Glu46Gln I-nutation shifted the ground state absorption maximum from 446 Co 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds, Arg52, which separates the active site from solvent in the ground state, was substituted by Ala, The smaller reef shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pK(d) = 8), Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle, The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pK(a) values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.