Proton-coupled
electron transfer reactions play critical roles
in many aspects of sensory phototransduction. In the case of flavoprotein
light sensors, reductive quenching of flavin excited states initiates
chemical and conformational changes that ultimately transmit light
signals to downstream targets. These reactions generally require neighboring
aromatic residues and proton-donating side chains for rapid and coordinated
electron and proton transfer to flavin. Although photoreduction of
flavoproteins can produce either the anionic (ASQ) or neutral semiquinone
(NSQ), the factors that favor one over the other are not well understood.
Here we employ a biologically active variant of the light-oxygen-voltage
(LOV) domain protein VVD devoid of the adduct-forming Cys residue
(VVD-III) to probe the mechanism of flavin photoreduction and protonation.
A series of isosteric and conservative residue replacements studied
by rate measurements, fluorescence quantum yields, FTIR difference
spectroscopy, and molecular dynamics simulations indicate that tyrosine
residues facilitate charge recombination reactions that limit sustained
flavin reduction, whereas methionine residues facilitate radical propagation
and quenching and also gate solvent access for flavin protonation.
Replacement of a single surface Met residue with Leu favors formation
of the ASQ over the NSQ and desensitizes photoreduction to oxidants.
In contrast, increasing site hydrophilicity by Gln substitution promotes
rapid NSQ formation and weakens the influence of the redox environment.
Overall, the photoreactivity of VVD-III can be understood in terms
of redundant electron donors, internal hole quenching, and coupled
proton transfer reactions that all depend upon protein conformation,
dynamics, and solvent penetration.