BLUF domain function does not require a metastable radical intermediate state

Lukacs, Andras, Brust, Richard, Haigney, Allison, Laptenok, Sergey P., Addison, Kiri, Gil, Agnieszka, Towrie, Michael, Greetham, Gregory M., Tonge, Peter J. and Meech, Stephen R. ORCID: https://orcid.org/0000-0001-5561-2782 (2014) BLUF domain function does not require a metastable radical intermediate state. Journal of the American Chemical Society, 136 (12). pp. 4605-4615. ISSN 0002-7863

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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.

Item Type:	Article
Additional Information:	ACS AuthorChoice - Terms of Use CC-BY
Faculty \ School:	Faculty of Science > School of Chemistry Faculty of Science
UEA Research Groups:	Faculty of Science > Research Groups > Physical and Analytical Chemistry (former - to 2017) Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Groups > Centre for Photonics and Quantum Science
Depositing User:	Pure Connector
Date Deposited:	09 Jul 2014 10:00
Last Modified:	10 Nov 2023 02:26
URI:	https://ueaeprints.uea.ac.uk/id/eprint/49047
DOI:	10.1021/ja4121082

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