Identification of the TyrOH^•+ Radical Cation in the Flavoenzyme TrmFO

Abstract

Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOH, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOH is formed in ∼1 ps by electron transfer to excited flavin and decays in ∼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates.

Short‐Lived Radical Intermediates in the Photochemistry of Glucose Oxidase

“…However, the spectrum in the visible associated with charge recombination was rather poorly reproduced with model spectra of radical intermediates available at the time. They show some reminiscence, by contrast, with the spectrum recently assigned to the FAD .− TyrOH .+ radical pair …”

“…Here, the scaling factor α was taken such that the scaled FAD .– minus FAD ox extinction spectrum (blue, Figure ) matches the bleaching in S(λ) (green, Figure ) in the blue part of the spectrum (<440 nm). Indeed, the obtained X 4ps (λ) spectrum has an absorption peak at ∼490 nm, as the spectrum assigned to TyrOH .+ in a TrmFO variant . We note there are also significant spectral differences, as will be discussed below, and the need of the subtraction procedures of Equations 1 and 2 yields some uncertainty in the precise shape of the band, but significant absorption in this spectral region is always required.…”

“…The time‐resolved fluorescence setup uses a Kerr gate and has been described before . The excitation pulse centered at 390 nm is obtained by frequency‐doubling, using a BBO crystal, part of the 780‐nm pulse from the Ti:sapphire laser/amplifier system (Quantronix Integra‐C) operating at 1 kHz.…”

A Molecular Mechanics Model for Flavins

“…The pK a of the N5 nitrogen of flavin is 8.3 pH units in the radical form . When bound to a protein and in the absence of a proton donor, the anionic radical form can exist for a significant period of time after the oxidized form of flavin is reduced by the transfer of one electron . Thus, this protonation state of the flavin radical was also parametrized in this work.…”

“…Flavins can also act in synergy with other cofactors such as iron–sulfur clusters, heme, molybdopterin, or thiamine diphosphate, increasing the diversity of reactions . Time‐resolved spectroscopy allowed gaining a wealth of information on the protein dynamics of flavoproteins, on timescales directly accessible by Molecular Dynamics (MD) simulations …”