NEW INSIGHTS INTO THE PHOTOCYCLE OF ECTOTHIORHODOSPIRA-HALOPHILA PHOTOACTIVE YELLOW PROTEIN - PHOTORECOVERY OF THE LONG-LIVED PHOTOBLEACHEDINTERMEDIATE IN THE MET100ALA MUTANT

There are previously two known intermediates (I-1 and I-2) in the room -temperature photocycle of the photoactive yellow protein (PYP) from E ctothiorhodospira halophila. The three-dimensional structures of groun d-state PYP and of I-2 have shown that Light-induced conformational ch anges are localized to the active site. Previous site-specific mutagen esis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala). We non report the construction a nd characterization of a mutant at a third active site position (Met10 0Ala) in order to establish the role of this residue in the photocycle . Met100Ala PYP has an absorption spectrum which is very similar to wi ld-type (WT) PYP, but exhibits very different kinetic properties, At p H 7.0, the light-induced bleaching reaction (I-2 formation) has a half -life <1 mu s and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 mu s and 140 ms for the same react ions in WT PYP. The slow rate of recovery from It for Met100Ala result s in the accumulation of the bleached intermediate even under room lig ht illumination. These results are qualitatively similar to what has b een observed with the Arg52Ala mutant of PYP, and with WT PYP in the p resence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein. The midpoint for guanidine denaturat ion confirms this, The slow recovery of I-2 in the Met100Ala mutant ha s allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 n m laser flash, with kinetics (half-life = 160 mu s; k = 6300 s(-1)) wh ich are 6 orders of magnitude faster than dark recovery. This implies that chromophore reisomerization limits the rate of conversion of I-2 to the ground state in PYP. Met100 is in van der Waals contact with th e chromophore in the I-2 state, and we suggest that the sulfur atom ca talyzes cis-trans isomerization in WT PYP.