Fig. 2: Spectroscopic and enzymatic characterization of DmF1 and DrF1.

a UV–vis absorbance spectra of DmF1 upon illumination with far-red light (800 nm, purple line) or red light (650 nm, red line). The dotted line denotes the pure Pfr spectrum calculated according to⁶⁹. All spectra were normalized to the absorbance at 700 nm after far-red light exposure. b As panel a but for DrF1. c The Pr→Pfr photoactivation kinetics of DmF1 (green) and DrF1 (orange) driven by red light (red flash), and the corresponding Pfr→Pr reversion kinetics under far-red light (purple flash) were monitored by the absorbance at 750 nm and evaluated according to single-exponential functions. d The dark recovery of DmF1 (green) and DrF1 (orange) after illumination with saturating red light was observed at 750 nm. For reference, the dashed green and orange lines denote the absorbance at 750 nm in the far-red illuminated spectra of DmF1 and DrF1 (see panels a, b). e When incubated in darkness (‘D’) in the presence of ATP, both DmF1 and DrF1 trigger an increase in phosphorylated FixJ (p-FixJ) as visualized by slower migration in a Phos-tag gel than the unphosphorylated form. By contrast, under red light (‘R’), FixJ remains unphosphorylated, as it does if no SHK is included in the reaction (‘-‘). Experiments were repeated three times with similar outcomes. f DmF1 and DrF1 were incubated in the presence of FixJ and a double-stranded DNA molecule containing the operator site for phospho-FixJ and a 5′-attached TAMRA fluorescence label (see Suppl. Fig. 6a). Following illumination with far-red light (to populate the Pr state, indicated by purple bar), the reaction was initiated by ATP addition, and the TAMRA fluorescence anisotropy was monitored over time. The signal rise indicates formation of phospho-FixJ and binding to the DNA. Illumination with red light (marked in red) leads to a decrease in signal, indicative of net phosphatase activity. Experiments were done in triplicate (n = 3) with similar outcomes (see Suppl. Fig. 6b, c). Source data are provided as a Source Data file.