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Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus

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Abstract

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor–acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

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Acknowledgements

B. Z. is supported by a China Scholarship Council PhD scholarship. Part of this work was performed using High Performance Computing resources from Grand Equipement National pour le Calcul Intensif [Centre de Calcul Recherche et Technologie/Centre Informatique National de l’Enseignement Supérieur/Institut du Développement et des Ressources en Informatique Scientifique] (Grant 2018-2019, project number A0020706913).

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Authors and Affiliations

  1. LOB, CNRS, INSERM, Ecole Polytechnique, Institut Polytechnique de Paris, 91128, Palaiseau Cedex, France

    Bo Zhuang, Lipsa Nag, Pierre Sournia, Anastasia Croitoru, Rivo Ramodiharilafy, Jean-Christophe Lambry, Hannu Myllykallio, Alexey Aleksandrov, Ursula Liebl & Marten H. Vos

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  1. Bo Zhuang
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Correspondence to Marten H. Vos.

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Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer - a topical issue in honour of Klaus Brettel.

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Zhuang, B., Nag, L., Sournia, P. et al. Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus. Photochem Photobiol Sci 20, 663–670 (2021). https://doi.org/10.1007/s43630-021-00052-8

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