The protein encoded by the gene slr1393 from the cyanobacterium Synechocystis sp. PCC6803 (Slr1393) is composed of three GAF domains, a PAS domain, and a histidine kinase motif. The third GAF domain (referred to as GAF3) was previously characterized as the sole domain in this protein, being able to carry phycocyanobilin (PCB) as the chromophore and to accomplish photochemistry. GAF3 shows photochromicity, and is able to switch between a red-absorbing parental state (GAF3R, λmax = 649 nm) and a greenabsorbing photoproduct state (GAF3G, λmax = 536 nm) upon appropriate irradiation. In this study we have determined the photochemical quantum yields for the interconversion between both forms using two methods: an “absolute” method and a reference-based control. The latter is a comparative procedure which exploits a well-characterized blue-light photoreceptor, YtvA from Bacillus subtilis, and the cyanobacterial phytochrome Cph1 as actinometers. The former is an ad hoc developed, four laser-based setup where two cw lasers provide the pump beams to induce photoswitching (red to green and green to red, respectively) and two cw lasers simultaneously monitor the appearance and disappearance of the two species. Interestingly, fit analysis of the recorded transient absorbance changes provided a quantum yield for the green → red conversion (≈0.3) at least three times larger than for the red → green conversion (≈0.08). These data are in agreement with the results from the comparative method documenting the usefulness of the ‘direct’ method developed here for quantum yields’ determination. The light-induced switching capability of this photochromic protein allowed measuring the kinetics of GAF3 immobilized on a glass plate, and within living, overexpressing Escherichia coli cells.
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