Thermochromatium tepidum photoactive yellow protein/bacteriophytochrome/diguanylate cyclase: characterization of the PYP domain

The purple phototrophic bacterium, Thermochromatium tepidum, contains a gene for a chimeric photoactive yellow protein/bacteriophytochrome/diguanylate cyclase (Ppd). We produced the Tc. tepidum PYP domain (Tt PYP) in Escherichia coli, and found that it has a wavelength maximum at 358 nm due to a Leu46 substitution of the color-tuning Glu46 found in the prototypic Halorhodospira halophila PYP (Hh PYP). However, the 358 nm dark-adapted state is in a pH-dependent equilibrium with a yellow species absorbing at 465 nm (pK(a) = 10.2). Following illumination at 358 nm, photocycle kinetics are characterized at pH 7.0 by a small bleach and red shift to what appears to be a long-lived cis intermediate (comparable to the I(2) intermediate in Hh PYP). The recovery to the dark-adapted state has a lifetime of approximately 4 min, which is approximately 1500 times slower than that for Hh PYP. However, when the Tt PYP is illuminated at pH values above 7.5, the light-induced difference spectrum indicates a pH-dependent equilibrium between the I(2) intermediate and a red-shifted 440 nm intermediate. This equilibrium could be responsible for the sigmoidal pH dependence of the recovery of the dark-adapted state (pK(a) = 8.8). In addition, the light-induced difference spectrum shows that, at pH values above 9.3, there is an apparent bleach near 490 nm superimposed on the 358 and 440 nm changes, which we ascribe to the equilibrium between the protonated and ionized dark-adapted forms. The L46E mutant of Tt PYP has a wavelength maximum at 446 nm, resembling wild-type Hh PYP. The kinetics of recovery of L46E following illumination with white light are slow (lifetime of 15 min at pH 7), but are comparable to those of wild-type Tt PYP. We conclude that Tt PYP is unique among the PYPs studied to date in that it has a photocycle initiated from a dark-adapted state with a protonated chromophore at physiological pH. However, it is kinetically most similar to Rhodocista centenaria PYP (Ppr) despite the very different absorption spectra due to the lack of E46.