The first molecular characterisation of blue- and red-light photoreceptors from Methylobacterium radiotolerans

Methylobacteria are facultative methylotrophic phytosymbionts of great industrial and agronomical interest, and are considered as opportunistic pathogens a health threat for humans. So far only few reports mention photoreceptors coding sequences in Methylobacteria genomes, but no investigation at the molecular level has been performed yet. We here present a comprehensive in silico research of potential photoreceptors in this bacterial phylum and report photophysical and photochemical characterisation of two representatives of the most wide-spread photoreceptor classes, a blue-light sensing LOV (Light, Oxygen, Voltage) protein and and red/far red light sensing BphP (biliverdin-binding bacterial phytochrome) from M. radiotolerans JCM 2831. Overall, both proteins undergo the expected light-triggered reactions, but peculiar features were also identifed. The LOV protein Mr4511 has an extremely long photocycle and lacks a tryptophan conserved in ca. 75% of LOV domains. Mutation I37V accelerates the photocycle by one order of magnitude, while the Q112W change underscores the ability of tryptophan in this position to perform efficient energy transfer to the flavin chromophore. Time-resolved photoacoustics experiments showed that Mr4511 has a higher triplet quantum yield than other LOV domains and that formation of the photoproduct results in a volume expansion, in sharp contrast to other LOV proteins. Mr4511 was found astonishingly resistant to denaturation by urea, still showing the light-triggered reactions after incubation in urea for more than 20 h. The phytochrome MrBphP1 exhibits the so far most red-shifted absorption maxima for its Pr- and Pfr forms (λmax = 707 nm, 764 nm for the Pr, Pfr-forms). The light-driven conversions in both directions occur with relatively high quantum yields of 0.2. Transient ns absorption spectroscopy (μs-ms time range) identifies the decay of the instantaneously formed lumi-intermediate, followed by only one additional intermediate before formation of the respective final photoproduct for Pr-to-Pfr or Pfr-to-Pr photoconversion, in contrast to other BphPs. The relatively simple photoconversion patterns suggest the absence of shunt pathways reported for other bacterial