Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in

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 level. 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 Gln, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 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 red 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 (pKa = 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 pKa 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.

A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum. Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain. Reconstitution experiments demonstrate that Ppr covalently attaches the blue light-absorbing chromophore p-hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP. Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light. Phylogenetic analysis demonstrates that R. centenum Ppr may be ancestral to cyanobacterial and plant phytochromes.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in