Green, red, near-infrared: biophysical investigations on bacterial bilin-binding photoreceptors

Bacterial photoreceptors binding open-chain tetrapyrroles (bilins) as chromophores are related to plant phytochromes (phy) as they are photochromic and the primary photochemistry consists of a Z/E isomerisation around the bilin 15=16 double bond. The chromophore is embedded in all cases within a so-called GAF domain with a typical α/β fold. Different to the canonical plant phys that invariably bind phytochromobilin and switch between a red and a far red absorbing form (R/FR), the bacterial bilin-photoreceptors exhibit a much wider variety of spectroscopic and functional properties, and bind diverse bilin chromophores, e.g. phycocyanobilin (PCB) and biliverdin (BV). In particular, isolated GAF domains of cyanobacteriochromes (CBCRs) take on great importance, because they are photochromic as standalone units. Beyond their intrinsic interest as light-sensing systems in prokaryots, these proteins show features, as a larger fluorescence quantum yield (F) and broader spectral ranges, that render them good candidates for biotechnological applications. Here we report steady-state and time-resolved spectroscopic measurements on selected bacterial bilin-photoreceptors: a. BV-binding phy from Pseudomonas strains with FR/NIR photochromism; b. PCB-binding GAF3 domain of the R/G (Red/Green) switching CBCR 1393 (Slr1393g3) from Synechocystis; c. GAF1 (R/FR) and GAF3 (R/Orange) domains of Anabaena 2699 (All2699). For CBCR GAF domains, both wild-type and mutated proteins, for which the dynamics of light-triggered reactions is altered, were analyzed. Within this pool of bacterial bilin-photoreceptors, a good correlation was found between F and fluorescence lifetimes. Nanosecond time-resolved absorption spectroscopy revealed the kinetics and spectral features of transient species after photoactivation. The most unusual behavior was found for the G-form of Slr1393g3: this form exhibits steady-state fluorescence heterogeneity and an up to now undescribed optical transient with a lifetime of ca. 60 ns at 20°C, upon green-light excitation. This transient was first uncovered by means of a photothermal method, but was now tracked by careful inspections of transient optical kinetic traces. Definitely, further investigations will be necessary to enlighten a possible link between the detected heterogeneity of Slr1393g3-G and this novel transient species.