BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

Andras Lukacs,Richard Brust,Sergey P Laptenok,Gregory M Greetham,Peter J Tonge,Stephen R Meech,Allison Haigney,Kiri Addison,Michael Towrie,Agnieszka Gil

doi:10.1021/ja4121082

Abstract

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered.

Highlights

Light-sensing proteins mediate the response of living systems to light
From a comparison with the transient infrared (TRIR) of FMN in buffer solution (Figure 2B) it is evident that on the nanosecond time scale the dAppABLUF spectrum is dominated by flavin ring localized vibrational modes, with negative peaks arising from depletion of the electronic ground state and positive peaks appearing during the excitation pulse arising from excited state modes
The primary processes in the blue light sensing using FAD (BLUF) domain have been investigated by ultrafast TRIR spectroscopy

Summary

Introduction

Light-sensing proteins mediate the response of living systems to light. In the most widely studied examples, rhodopsins, phytochromes, and photoactive yellow protein, the primary process involves an excited state isomerization reaction.[1,2] Relatively recently a range of blue-light-sensing flavoproteins have been discovered and shown to be widespread, occurring in animals, plants, fungi, and bacteria.[3−5] Three separate classes have been identified: photolyase/cryptochromes; lightoxygen-voltage (LOV) domain proteins; blue light sensing using FAD (BLUF) domain proteins. The mechanism of operation of these photoactive flavoproteins is a topic of intense experimental and theoretical investigation.[6,7] In the DNA repair enzyme, photolyase, a change in oxidation state of the flavin is observed, while in the LOV domain a reaction of the triplet state of the flavin with an adjacent cysteine is the primary mechanism.[8−11]

Methods

Results

Conclusion