Oral Presentation 18th International Congress on Photobiology 2024

Photoswitching of Flavin–Inhibitor Complexes in Flavoenzymes (#16)

Bo Zhuang 1 , Guangliu Ran 2 , Marten Vos 3 , Wenkai Zhang 2 , Feng Gai 1
  1. College of Chemistry and Molecular Engineering, Peking University, Beijing, China
  2. Department of Physics, Beijing Normal University, Beijing, Beijing, China
  3. Laboratoire d'optique et biosciences, École Polytechnique, Palaiseau, France

The vast majority of flavoenzymes, including monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX), perform non-light-driven physiological functions.[1] However, the involvement of flavin cofactors in photoinduced reactions is widespread in nature.[2] MSOX and MTOX both harbor an oxidized flavin adenine dinucleotide (FADox) cofactor, and catalyze the oxidative demethylation of sarcosine and N-methyltryptophan, respectively. Methylthioacetate (MTA) and methylselenoacetate (MSeA) are substrate analog inhibitors of MSOX that form complexes with MSOX, exhibiting intense absorption bands over the whole visible spectral range due to flavin−MXA (X = T, Se) charge-transfer (CT) interactions (Fig. 1a). Based on femtosecond transient absorption (TA) measurements, we show that when the MSOX:MXA complexes are photoexcited, the CT interactions disappear during a barrierless reaction in ca. 300 fs with a high quantum yield (Fig. 1d). The initial complex subsequently geminately reforms in a few nanoseconds near room temperature in a thermally activated way (Fig. 1b).[3] In the experimental crystal structure of MSeA-containing MSOX, the ligand is bound to the protein in two discrete conformations (Fig. 1c; Conf 1 vs. Conf 2). Considering the timescales of the reactions, it is highly plausible that on the molecular level, the switching of the FADox:MXA complexes involves the isomerization of MXA between Conf 1 and Conf 2.[4]

                                                                                                      667c320398f0c-iupa_s.png

Fig. 1. (a) Spectra of the FADox:MXA  complexes in MSOX. (b) Photoswitching of the FADox:MTA complex in MSOX at 77K. (c) Active-site structures of MSOX and MTOX containing MSeA in two conformations. (d) TA kinetics of the MSOX and MTOX variants complexed with MSeA pumped at 550 nm and probed at ca. 600 nm.

By contrast, in MTOX, which can also bind MSeA to form a CT complex, we did not observe efficient photoswitching of the FADox:MSeA complex upon excitation, with a slower forward switching (ca. 700 fs) and a much faster back recovery (ca. 140 ps). As a close homologue of MSOX, MTOX has an active site that highly resembles that of MSOX in structure, with the exception that a methionine residue, Met245, in MSOX, closely interacting with the MSeA ligand in Conf 2, is replaced by a threonine (Thr239) in MTOX, located farther away from the ligand (Fig. 1c). This indicates that the presence of the nearby Met245 is a prerequisite for efficient photoswitching, which is corroborated by the experiment on the T239M mutant of MTOX, where the threonine-to-methionine mutation significantly activates the photoswitching, slowing down the back recovery to a nanosecond timescale (Fig. 1d). Molecular dynamics simulations and quantum chemical calculations provide detailed insights into the interactions between FADox and MXA in the protein active sites, and demonstrate the effects of Met245 on the conformations of MXA and the energetics involved.[5] Conformational photochemical processes in CT complexes may be further explored for novel photocatalytic and photoswitching applications of flavoproteins.

  1. Ghisla, S.; Massey, V., Eur. J. Biochem. 1989, 181, 1–17.
  2. Zhuang, B.; Liebl, U.; Vos, M. H., J. Phys. Chem. B 2022, 126, 3199–3207.
  3. Zhuang, B.; Vos, M. H., J. Am. Chem. Soc. 2022, 144, 11569–11573.
  4. Wagner, M. A.; Trickey, P.; Che, Z. W.; Mathews, F. S.; Jorns, M. S. Biochemistry 2000, 39, 8813–8824.
  5. Zhuang, B.; Ran, G.; Vos, M. H.; Zhang W.; Gai, F., in preparation.