BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

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.

Electron transfer quenching in light adapted and mutant forms of the AppA BLUF domain

“…The previously studied (photoinactive) mutant Y21W also showed rapid and efficient electron transfer and clear intermediate state kinetics; thus, in that case Trp is unambiguously the electron donor. 41 The rate of electron transfer in Y21W was much higher than for the present Y21 mutants, which is consistent with W21 being located close to the isoalloxazine ring. Interestingly along with the expected radical isoalloxazine feature at 1531 cm À1 , Y21W showed an additional intense sigmoidal transient/bleach feature about 1630 cm À1 in both the experimental spectra and the EADS recovered from global analysis.…”

“…Interestingly along with the expected radical isoalloxazine feature at 1531 cm À1 , Y21W showed an additional intense sigmoidal transient/bleach feature about 1630 cm À1 in both the experimental spectra and the EADS recovered from global analysis. 41 This 1630 cm À1 feature is also evident in the EADS of Y21S (Fig. 4a) and other Y21 mutants, but is of lower amplitude than for Y21W.…”

“…3b. The separation between the two is apparent, whereas for dAppA BLUF the traces overlapped, 41 suggesting the excited state decay and ground state recovery match, consistent with a short lived (or absent) intermediate in dAppA BLUF .…”

“…Using TRIR we have previously identied marker modes for the excited state decay and ground state recovery. 41 Thus these can be t separately, and any difference between them suggests the formation of a metastable intermediate, which should also be visible in TRIR spectra (for example in the case of electron transfer, the radical state of the isoalloxazine ring). Thus transient data at the two characteristic wavenumbers were analysed separately.…”

“…Recently Lukacs et al presented a detailed study of the TRIR of three BLUF domains and contrasted the results with model spectra of radical isoalloxazines and with avoproteins which unambiguously show electron transfer reactions. 41 Although radical states are observed in some cases there was no correlation between BLUF photoactivity and electron transfer. Further, unnatural amino acid substitutions were used to articially modulate the redox potential of the putative Y21 donor, but the results were not consistent with a simple electron transfer reaction.…”