Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors

• Published in: The Journal of biological chemistry 2012-09, Vol.287 (38), p.31725-31738
• Main Authors: Mathes, Tilo, van Stokkum, Ivo H.M., Stierl, Manuela, Kennis, John T.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Crystallography, X-Ray - methods; Electron Transfer; Electron Transport; Escherichia coli - metabolism; Flavin; Flavins - chemistry; Kinetics; Light; Molecular Biophysics; Molecular Conformation; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oligonucleotides - genetics; Oxidation-Reduction; Photochemistry - methods; Photoreceptors; Photoreceptors, Microbial - chemistry; Redox; Redox Potential; Spectrophotometry - methods; Spectroscopy; Synechocystis - metabolism; Tyrosine - chemistry; Ultrafast Spectroscopy
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.391896

Photoinduced electron transfer in biological systems, especially in proteins, is a highly intriguing matter. Its mechanistic details cannot be addressed by structural data obtained by crystallography alone because this provides only static information on a given redox system. In combination with transient spectroscopy and site-directed manipulation of the protein, however, a dynamic molecular picture of the ET process may be obtained. In BLUF (blue light sensors using FAD) photoreceptors, proton-coupled electron transfer between a tyrosine and the flavin cofactor is the key reaction to switch from a dark-adapted to a light-adapted state, which corresponds to the biological signaling state. Particularly puzzling is the fact that, although the various naturally occurring BLUF domains show little difference in the amino acid composition of the flavin binding pocket, the reaction rates of the forward reaction differ quite largely from a few ps up to several hundred ps. In this study, we modified the redox potential of the flavin/tyrosine redox pair by site-directed mutagenesis close to the flavin C2 carbonyl and fluorination of the tyrosine, respectively. We provide information on how changes in the redox potential of either reaction partner significantly influence photoinduced proton-coupled electron transfer. The altered redox potentials allowed us furthermore to experimentally describe an excited state charge transfer intermediately prior to electron transfer in the BLUF photocycle. Additionally, we show that the electron transfer rate directly correlates with the quantum yield of signaling state formation. Background: Proton-coupled electron transfer is the key step in BLUF photoactivation. Results: Redox modulation of flavin and tyrosine determines electron transfer rates and signaling efficiency and reveals a new photocycle intermediate. Conclusion: Partial charge transfer from tyrosine to flavin takes place prior to full electron transfer. Significance: Mechanistic details of protein-modulated electron transfer processes are crucial to understand biological proton-coupled electron transfer reactions.