Propensity for Proton Relay and Electrostatic Impact of Protein Reorganization in Slr1694 BLUF Photoreceptor

Abstract

Photoreceptor proteins play a vital role in a wide range of light-regulated processes. The formation of the light-adapted state of blue light using flavin (BLUF) photoreceptors is thought to involve rearrangements of hydrogen-bonding networks upon photoexcitation. Free energy simulations with partial charges corresponding to relevant ground and excited states of the Slr1694 BLUF domain characterize conformations prior to and following photoexcitation. The simulations indicate that Trp91 is thermodynamically favored to be in the active site, although it is also able to sample conformations outside the active site. For experimentally observed conformations of Trp91, Gln50 is thermodynamically favored to be oriented for a proton relay bridging Tyr8 and the flavin. When Trp91 is rotated such that it can donate a hydrogen bond to Gln50, as observed in other BLUF domains, the proton relay is not thermodynamically favored in the ground state, providing a possible explanation for the relatively fast photocycle of the Slr1694 BLUF domain. Photoexcitation to the locally excited (LE) state of the flavin induces the formation of the proton relay if it is not already formed. Electrostatically embedded time-dependent density functional theory calculations indicate that the proton relay reduces the energy gap between the LE state and the charge-transfer (CT) state associated with electron transfer from Tyr8 to the flavin. Although the CT state is higher in energy than the LE state prior to photoexcitation, the protein environment can reorganize in a manner that stabilizes the CT state so that it is lower than the LE state, enabling the LE to CT state transition. An electrostatic analysis identifies motions of individual residues, such as Arg65, that stabilize electron transfer from Tyr8 to the flavin. These conformational changes facilitate the critical proton-coupled electron transfer reaction in the BLUF photocycle.

Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems

“…In the case of PixD, we observed a sequential radical formation: first, Y8 donated an electron to the flavin and FAD ⋅− radical formed, neutral flavin radical was formed concomitantly in ~ 100 ps [15]. Based on theoretical calculations, in the case of PixD protonation of the flavin happens sequentially: after excitation the tyrosine gives a proton to an adjacent glutamine which finally protonates the flavin, stabilizing the neutral radical state [46,47]. In the case of PaPB, a BLUF protein found in the purple bacterium Rhodopseudomonas palustris, FADH ⋅ was formed directly-as in the case of the AppA W104Y mutant-via a proton-coupled electron transfer process [38].…”

Site-Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

“…[33][34] Recent free energy simulations coupled with electronic structure calculations suggest a dynamic picture of the flavin binding fold, with Trpin/Metout and Trpout/Metin in thermal equilibrium at physiological temperatures, modulating the probability of Tyr to flavin electron transfer and H-bond reorganization. [35][36][37] Other calculations suggest a dynamic binding pocket, with spontaneous glutamine rotation occurring on the time scale of the photoreaction. 38 Here we site specifically probe the dynamic response of AppABLUF and PixD to electronic excitation, combining µOD sensitivity ultrafast TRIR with UAA substitution at the critical Met and Trp positions.…”

Frontiers in Multiscale Modeling of Photoreceptor Proteins

“…Path CVs have been successfully used in blue-light using flavin (BLUF) photoreceptors, to efficiently extract mechanistic details and free energies (121,122). Other path-based methods that do not require biasing, such as transition path sampling (TPS) (123), have also been used in PYP photoreceptors (124), and similar principles have been applied to rhodopsin by tracking the time evolution of an excited-state population (125).…”