Formation of Interacting Spins on Flavosemiquinone and Tyrosine Radical in Photoreaction of a Blue Light Sensor BLUF Protein TePixD

被引:27
作者
Nagai, Hiroko [1 ]
Fukushima, Yoshimasa [1 ]
Okajima, Koji [2 ,3 ]
Ikeuchi, Masahiko [2 ]
Mino, Hiroyuki [1 ]
机构
[1] Nagoya Univ, Grad Sch Sci, Div Mat Sci Phys, Chikusa Ku, Nagoya, Aichi 4648602, Japan
[2] Univ Tokyo, Grad Sch Arts & Sci, Dept Life Sci Biol, Meguro Ku, Tokyo 1538902, Japan
[3] Osaka Prefecture Univ, Grad Sch Sci, Dept Biol Sci, Osaka 5998531, Japan
关键词
D O I
10.1021/bi8010187
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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(center dot). The FADH(center dot) 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(center dot)-Y8(center dot) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 angstrom 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(center dot)-Y8(center dot) 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(center dot)-Y8(center dot) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.
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页码:12574 / 12582
页数:9
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