Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

Agnieszka A. Gil; Sergey P. Laptenok; James N. Iuliano; Andras Lukacs; Anil Verma; Christopher R. Hall; Grace E. Yoon; Richard Brust; Gregory M. Greetham; Michael Towrie; Jarrod B. French; Stephen R. Meech; Peter J. Tonge

doi:10.1021/jacs.7b07849

Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

Agnieszka A. Gil, Sergey P. Laptenok, James N. Iuliano, Andras Lukacs, Anil Verma, Christopher R. Hall, Grace E. Yoon, Richard Brust, Gregory M. Greetham, Michael Towrie, Jarrod B. French, Stephen R. Meech, Peter J. Tonge

Research output: Contribution to journal › Article › peer-review

39 Scopus citations

Abstract

The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppA_BLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pK_a and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pK_a led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppA_BLUF, modulation in the Tyr (Y8) pK_a has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pK_a by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pK_a on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA_BLUF, despite their sharing highly conserved FAD binding architectures.

Original language	English (US)
Pages (from-to)	14638-14648
Number of pages	11
Journal	Journal of the American Chemical Society
Volume	139
Issue number	41
DOIs	https://doi.org/10.1021/jacs.7b07849
State	Published - Oct 18 2017
Externally published	Yes

Bibliographical note

Funding Information:
Funded by the EPSRC (EP/G002916 and EP/N033647/1 to S.R.M.) and NSF (CHE-1223819 to P.J.T.). We are grateful to STFC for access to the ULTRA laser facility. We are grateful to Professor Ray Owens for assistance in protein preparation and access to the Oxford Protein Production Facility, UK, and to Arthur Makarenko at CSHL for assistance with the mass spectrometry analysis. J.I. was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). A.L. is a Bolyai Janos Research Fellow and was supported by OTKA NN113090.

Publisher Copyright:
© 2017 American Chemical Society.

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10.1021/jacs.7b07849

http://hdl.handle.net/10754/625460

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Gil, A. A., Laptenok, S. P., Iuliano, J. N., Lukacs, A., Verma, A., Hall, C. R., Yoon, G. E., Brust, R., Greetham, G. M., Towrie, M., French, J. B., Meech, S. R., & Tonge, P. J. (2017). Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues. Journal of the American Chemical Society, 139(41), 14638-14648. https://doi.org/10.1021/jacs.7b07849

Gil, AA, Laptenok, SP, Iuliano, JN, Lukacs, A, Verma, A, Hall, CR, Yoon, GE, Brust, R, Greetham, GM, Towrie, M, French, JB, Meech, SR & Tonge, PJ 2017, 'Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues', Journal of the American Chemical Society, vol. 139, no. 41, pp. 14638-14648. https://doi.org/10.1021/jacs.7b07849

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title = "Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues",

abstract = "The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.",

author = "Gil, {Agnieszka A.} and Laptenok, {Sergey P.} and Iuliano, {James N.} and Andras Lukacs and Anil Verma and Hall, {Christopher R.} and Yoon, {Grace E.} and Richard Brust and Greetham, {Gregory M.} and Michael Towrie and French, {Jarrod B.} and Meech, {Stephen R.} and Tonge, {Peter J.}",

note = "Funding Information: Funded by the EPSRC (EP/G002916 and EP/N033647/1 to S.R.M.) and NSF (CHE-1223819 to P.J.T.). We are grateful to STFC for access to the ULTRA laser facility. We are grateful to Professor Ray Owens for assistance in protein preparation and access to the Oxford Protein Production Facility, UK, and to Arthur Makarenko at CSHL for assistance with the mass spectrometry analysis. J.I. was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). A.L. is a Bolyai Janos Research Fellow and was supported by OTKA NN113090. Publisher Copyright: {\textcopyright} 2017 American Chemical Society.",

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doi = "10.1021/jacs.7b07849",

language = "English (US)",

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T1 - Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

AU - Gil, Agnieszka A.

AU - Laptenok, Sergey P.

AU - Iuliano, James N.

AU - Lukacs, Andras

AU - Verma, Anil

AU - Hall, Christopher R.

AU - Yoon, Grace E.

AU - Brust, Richard

AU - Greetham, Gregory M.

AU - Towrie, Michael

AU - French, Jarrod B.

AU - Meech, Stephen R.

AU - Tonge, Peter J.

N1 - Funding Information: Funded by the EPSRC (EP/G002916 and EP/N033647/1 to S.R.M.) and NSF (CHE-1223819 to P.J.T.). We are grateful to STFC for access to the ULTRA laser facility. We are grateful to Professor Ray Owens for assistance in protein preparation and access to the Oxford Protein Production Facility, UK, and to Arthur Makarenko at CSHL for assistance with the mass spectrometry analysis. J.I. was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). A.L. is a Bolyai Janos Research Fellow and was supported by OTKA NN113090. Publisher Copyright: © 2017 American Chemical Society.

PY - 2017/10/18

Y1 - 2017/10/18

N2 - The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.

AB - The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.

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Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

Abstract

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