On the Role of Aromatic Side Chains in the Photoactivation of BLUF Domains
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- 作者：Magdalena Gauden ; Jeffrey S. Grinstead ; Wouter Laan ; Ivo H. M. van Stokkum ; Marcela Avila-Perez ; K. C. Toh ; Rolf Boelens ; Robert Kaptein ; Rienk van Grondelle ; Klaas J. Hellingwerf ; John T. M. Kennis
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：June 26, 2007
- 年：2007
- 卷：46
- 期：25
- 页码：7405 - 7415
- 全文大小：415K
- 年卷期：v.46,no.25(June 26, 2007)
- ISSN：1520-4995
文摘
BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensingreceptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which arein close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, isinvestigated through the introduction of several amino acid substitutions at these positions. NMRspectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was notsignificantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorptionspectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In theY21 mutants, FADH
-W radical pairs are transiently formed on a ps time scale and recombine to theground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wildtype AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant,all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves byintersystem crossing to the triplet state. Our results indicate that two competing light-driven electron-transfer pathways are available in BLUF domains: one productive pathway that involves electron transferfrom the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathwayfrom the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of thesignaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domainswhere signaling state formation proceeds via light-driven electron and proton transfer from the conservedtyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.
-W radical pairs are transiently formed on a ps time scale and recombine to theground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wildtype AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant,all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves byintersystem crossing to the triplet state. Our results indicate that two competing light-driven electron-transfer pathways are available in BLUF domains: one productive pathway that involves electron transferfrom the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathwayfrom the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of thesignaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domainswhere signaling state formation proceeds via light-driven electron and proton transfer from the conservedtyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.