| Autor: |
Gil AA, Laptenok SP; School of Chemistry, University of East Anglia , Norwich Research Park, Norwich NR4 7TJ, U.K., Iuliano JN, Lukacs A; Department of Biophysics, Medical School, University of Pecs , Pecs H-7622, Hungary., Verma A; Central Laser Facility, Harwell Science and Innovation Campus , Didcot, Oxon OX11 0QX, U.K., Hall CR; School of Chemistry, University of East Anglia , Norwich Research Park, Norwich NR4 7TJ, U.K., Yoon GE, Brust R, Greetham GM; Central Laser Facility, Harwell Science and Innovation Campus , Didcot, Oxon OX11 0QX, U.K., Towrie M; Central Laser Facility, Harwell Science and Innovation Campus , Didcot, Oxon OX11 0QX, U.K., French JB, Meech SR; School of Chemistry, University of East Anglia , Norwich Research Park, Norwich NR4 7TJ, U.K., Tonge PJ |
| Abstrakt: |
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. |
