Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Benita Kopka,Marco Bocola,Kathrin Magerl,Ulrich Schwaneberg,Katrin Röllen,Ulrich Krauss,Bernhard Dick,Mehdi D Davari,Anton Savitsky,Karl-Erich Jaeger

doi:10.1038/s41598-017-13420-1

Abstract

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Highlights

Flavin-binding proteins are ubiquitously distributed throughout all kingdoms of life, playing important roles either as redox catalysts[1,2] or molecular sensors for redox potential, oxygen or blue light[3,4,5]
The authors demonstrated that the accumulation of the neutral flavin semiquinone radical FMNH●, and flavin N5 protonation, is sufficient to elicit a functional response in photoreceptor variants containing an alanine instead of the photoactive cysteine, with reduced magnitude compared to the respective wild-type proteins[17]
LOV proteins in which the photoactive cysteine has been substituted for alanine cannot undergo adduct formation and show an intense cyan-green fluorescence resulting from excitation of the bound flavin chromophore[13,25,27]

Summary

Introduction

Flavin-binding proteins are ubiquitously distributed throughout all kingdoms of life, playing important roles either as redox catalysts[1,2] or molecular sensors for redox potential, oxygen or blue light[3,4,5] The latter function is intricately linked to the oxidation state and photochemistry of the bound flavin molecule. The authors demonstrated that the accumulation of the neutral flavin semiquinone radical FMNH●, and flavin N5 protonation, is sufficient to elicit a functional response in photoreceptor variants containing an alanine instead of the photoactive cysteine, with reduced magnitude compared to the respective wild-type proteins[17]. The residues which act as proton/electron donor in the photoreduction process remain largely elusive

Methods

Results

Conclusion