Photoreactions of Tyr8- and Gln50-mutated BLUF domains of the PixD protein of Thermosynechococcus elongatus BP-1: photoconversion at low temperature without Tyr8.
We studied the photoreaction of a blue-light sensor PixD protein of Thermosynechococcus elongatus that has the blue-light-using flavin (BLUF) domain. The Tyr8 and Gln50 residues of the protein were modified to phenylalanine, alanine, or asparagine (Y8F, Y8A, Q50N, and Q50A) by site-directed mutagenesis. The following results were obtained. (1) At room temperature, blue-light illumination induced the red shift of the absorption bands of flavin in the wild-type (WT) protein but not in the Y8F, Y8A, Q50A, and Q50N mutant proteins, as reported [Okajima, K., et al. (2006) J. Mol. Biol. 363, 10-18]. (2) At 80 K, neither the Q50N nor the Q50A mutant protein accumulated the red-shifted form. (3) At 80 K, the Y8F protein photoaccumulated the red-shifted forms to an extent that was half that in the WT protein at a 43-fold slower rate, and the Y8A protein to the one-fourth the extent at a 137-fold slower rate. (4) The red-shifted form in the Y8F protein was stable below 240 K and became unstable above 240 K in the dark. (5) The illumination of the Y8F protein at 150 K accumulated the red-shifted form at the beginning, and the prolonged illumination accumulated the flavin anions by the secondary photoreaction. (6) The results indicate that Tyr8 is not indispensable for the accumulation of the red-shifted form at least at 80 K. (7) Photoconversion mechanisms in the WT and Tyr8-mutated proteins are discussed in relation to the schemes with and without the electron transfer between Tyr8 and flavin in the first step of the photoconversion.
Fukushima
Yoshimasa
Y
Division of Material Science (Physics), Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan.
Murai
Yoshiya
Y
Okajima
Koji
K
Ikeuchi
Masahiko
M
Itoh
Shigeru
S
D000042:Absorption; D000838:Anions; D001426:Bacterial Proteins / Q000737:chemistry*; D000458:Cyanobacteria / Q000737:chemistry* / Q000528:radiation effects; D005182:Flavin-Adenine Dinucleotide; D005973:Glutamine / Q000235:genetics*; D006860:Hydrogen Bonding / Q000528:radiation effects; D008027:Light; D050505:Mutant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000235:genetics*
D000838:Anions; D001426:Bacterial Proteins; D050505:Mutant Proteins; D005973:Glutamine; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine