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

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

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


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 by the introduction of fluorotyrosine (F-Tyr) analogs 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 4,000-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, 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 AppA, despite their sharing highly conserved FAD binding architectures.
Original languageEnglish (US)
Pages (from-to)14638-14648
Number of pages11
JournalJournal of the American Chemical Society
Issue number41
StatePublished - Oct 5 2017

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: Funded by the EPSRC (EP/G002916 to SRM) and NSF (CHE-1223819 to PJT). 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. JI was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). AL is a Bolyai Janos Research Fellow and was supported by OTKA NN113090.


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