New Insights into the Photocycle of Ectothiorhodospira halophila Photoactive Yellow Protein:  Photorecovery of the Long-Lived Photobleached Intermediate in the Met100Ala Mutant

Abstract

There are previously two known intermediates (I1 and I2) in the room-temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The three-dimensional structures of ground-state PYP and of I2 have shown that light-induced conformational changes are localized to the active site. Previous site-specific mutagenesis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala). We now report the construction and characterization of a mutant at a third active site position (Met100Ala) in order to establish the role of this residue in the photocycle. Met100Ala PYP has an absorption spectrum which is very similar to wild-type (WT) PYP, but exhibits very different kinetic properties. At pH 7.0, the light-induced bleaching reaction (I2 formation) has a half-life <1 microseconds and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 microseconds and 140 ms for the same reactions in WT PYP. The slow rate of recovery from I2 for Met100Ala results in the accumulation of the bleached intermediate even under room light illumination. These results are qualitatively similar to what has been observed with the Arg52Ala mutant of PYP, and with WT PYP in the presence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein. The midpoint for guanidine denaturation confirms this. The slow recovery of I2 in the Met100Ala mutant has allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 nm laser flash, with kinetics (half-life = 160 microseconds; k = 6300 s-1) which are 6 orders of magnitude faster than dark recovery. This implies that chromophore reisomerization limits the rate of conversion of I2 to the ground state in PYP. Met100 is in van der Waals contact with the chromophore in the I2 state, and we suggest that the sulfur atom catalyzes cis-trans isomerization in WT PYP.

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

“…4A). It does not have an extra absorption peak at 350 nm, such as those observed in E-PYP Met-100 mutants, arising from a population of protonated trans chromophore because of destabilization of the chromophore binding pocket (27,44). Ppr-PYP also maintains the slow photocycle kinetics of full-length Ppr relative to E-PYP in solution (data not shown).…”

“…In the distal hydrogen-bonding network, Tyr-42 and Glu-46 are conserved in all PYPs, but not Thr-50, which is a Ser in Ppr-PYP. Residue Met-100, thought to catalyze chromophore reisomerization in E-PYP (27), is not highly conserved in PYPs, but is found in Ppr-PYP.…”

“…Structural changes in the ␤4-␤5 and ␣3-␣4 loops would be expected to affect photocycle dynamics because of the presence of critical residues in those regions (26,27). As in E-PYP, the ␤4-␤5 loop is flexible in Ppr-PYP, evidenced by high B factors in this region in chains A and B and its disorder in chain C. The disorder of the ␤4-␤5 loop of chain C is caused by a weak packing interaction between it and the N-terminal helices of chain A, which are similarly disordered.…”

“…Mutations of this residue slow the rate of decay from I 2 to the ground state by two to three orders of magnitude (27,44). Barring the access of Met-100 to the chromophore and changing the stability of the chromophore binding pocket by radically altering the conformation of the ␤4-␤5 loop would be expected to slow the photocycle considerably.…”

“…A R. centenum Ppr knockout showed no defects in phototaxis but had lower levels of expression of chalcone synthase, an enzyme involved in photoprotection through flavonoid biosynthesis (25). Although the PYP domain of Ppr (Ppr-PYP) is 45% identical in sequence to E-PYP and has similar or identical residues to all those suggested to be critical to absorption spectra and photocycle dynamics in E-PYP (26,27), Ppr-PYP has a considerably slower photocycle than E-PYP (25). Furthermore, as Ppr-PYP controls the activity of a histidine kinase, its study affords not only the opportunity to understand the signaling mechanism of PYPs, but of the entire class of PAS-domain-regulated sensor histidine kinases, which are ubiquitous in bacteria (28).…”