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-se nsing protein interact to create a light cycle, we performed time-reso lved spectroscopy on site-directed mutants of photoactive yellow prote in (PYP), Recently determined crystallographic structures of PYP in th e ground and colorless I2 states allowed us to design mutants and to s tudy their photosensing properties at the atomic revel. We developed a system for rapid mutagenesis and heterologous bacterial expression fo r PYP apoprotein and generated holoprotein through formation of a cova lent 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 i n the ground state, The Glu46Gln I-nutation shifted the ground state a bsorption maximum from 446 Co 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds, Arg52, which s eparates the active site from solvent in the ground state, was substit uted 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 int eresting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu 46Gln PYP in response to a change in pH from pH 5 to 10 (pK(d) = 8), P rompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle, The rate of color loss de creased about 3-fold with increasing pH from pH 5 to 10. The rate of r ecovery 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 r ate 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 ch anges with pH demonstrate that both phases in PYP's light cycle are ac tively controlled by the protein component.