The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides.
The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), 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 degrees 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 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 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 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) 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.
Haker
Andrea
A
Laboratory for Microbiology, Swammerdam Institute for Life Sciences, BioCentrum, University of Amsterdam, Nieuwe Achtergracht 166, The Netherlands.
Hendriks
Johnny
J
van Stokkum
Ivo H M
IH
Heberle
Joachim
J
Hellingwerf
Klaas J
KJ
Crielaard
Wim
W
Gensch
Thomas
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D008958:Models, Molecular; D020283:Photoreceptors, Microbial / Q000737:chemistry / Q000502:physiology*; D010788:Photosynthesis; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria