The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

Abstract

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP360) and 446 nm (R-PYP446), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 °C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 μs, and 17 μs. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB360, λmax = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 °C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP360 with a UV-flash (330 ± 50 nm) a species with a difference absorption maximum at ∼435 nm is observed that returns to R-PYP360 on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP360 and R-PYP446 differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.