Formation of interacting spins on flavosemiquinone and tyrosine radical in photoreaction of a blue light sensor BLUF protein TePixD.
Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.
Nagai
Hiroko
H
Division of Material Science (Physics), Graduate School of Science, Nagoya University, Furocho, Chikusa, Nagoya 464-8602, Japan.
Fukushima
Yoshimasa
Y
Okajima
Koji
K
Ikeuchi
Masahiko
M
Mino
Hiroyuki
H
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000458:Cyanobacteria; D003624:Darkness; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009154:Mutation; D055668:Photochemical Processes; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D001426:Bacterial Proteins; D011809:Quinones; C001636:flavosemiquinone; D014443:Tyrosine