Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO

Abstract

Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π–π stacking interaction, involving a conserved tyrosine (Y₃₄₆ in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C₅₃). Here, the interaction between FAD and Y₃₄₆ has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y₃₄₆ mutants by ultrafast transient absorption spectroscopy. In C53A, the excited FAD very rapidly (0.43 ps) abstracts an electron from Y₃₄₆, yielding the FAD˙⁻/Y₃₄₆OH˙⁺ radical pair, while relaxation of the local environment (1.3 ps) of the excited flavin produces a slight Stokes shift of its stimulated emission band. The radical pair then decays via charge recombination, mostly in 3–4 ps, without any deprotonation of the Y₃₄₆OH˙⁺ radical. Presumably, the H-bond between Y₃₄₆ and the amide group of C₅₃ increases the pK_a of Y₃₄₆OH˙⁺ and slows down its deprotonation. The dynamics of WT BsTrmFO shows additional slow decay components (43 and 700 ps), absent in the C53A mutant, assigned to excited FAD_ox populations not undergoing fast photoreduction. Their presence is likely due to a more flexible structure of the WT protein, favored by the presence of C₅₃. Interestingly, mutations of Y₃₄₆ canceling its electron donating character lead to multiple slower quenching channels in the ps–ns regime. These channels are proposed to be due to electron abstraction either (i) from the adenine moiety of FAD, a distribution of the isoalloxazine–adenine distance in the absence of Y₃₄₆ explaining the multiexponential decay, or (ii) from the W₂₈₆ residue, possibly accounting for one of the decays. This work supports the idea that H-bond-assisted π–π stacking controls TrmFO's active site dynamics, required for competent orientation of the reactive centers during catalysis.