Broadband fluorescence upconversion (FLUPS) and transient absorption spectroscopy (TAS)

All measurements were performed in 1 or 0.2 mm quartz cuvettes. The samples were prepared as described above at a complex concentration of 250 or 625 µM in 125 mM KCl, 40 mM HEPES pH 7.5, and 5 mM MgCl₂. Both spectrometers, the broadband fluorescence upconversion, and the transient absorption spectrometer, were driven by a chirped pulse amplified femtosecond laser “Solstice” from Newport-Spectra with a fundamental wavelength of 800 nm, a pulse width of 100 fs, and a repetition rate of 1 kHz.

The FLUPS is a commercial available spectrometer from LIOPTEC which can simultaneously measure 395–850 nm fluorescence with an intrinsic resolution of 0.9 nm (303–516 nm upconverted, intrinsic resolution 0.42 nm). The maximum delay between pump and gate pulse can be up to 1.500 ps. To generate the pump wavelength 405 nm for the FLUPS we used the output from a non collinear parametric amplifier which was set to 810 nm. The 810 nm were guided through a BBO crystal to generate the second harmonic at 405 nm. We used an 800 nm broadband bandpass filter to eliminate the residual 810 nm. The pump pulse energy was 50 nJ and the gate pulse energy was 30 uJ at 1280 nm. For more details on the setup see ref. ⁶⁴.

The pump pulse from the TAS was generated with a traveling wave oscillating parametric amplifier “TOPAS-C” from Light Conversion. The pump pulse energy was set to 40 nJ. The white light was generated with a moving CaF₂ crystal with some filters to achieve a probe light from 400 to 800 nm. The maximum delay between pump and probe was 8 ns. For a more detailed description of the setup see ref. ⁶⁵.

For both types of measurement we performed a global analysis of the data using GLOTARAN software⁶⁶. This analysis takes the white light dispersion and, in case of the TA measurements, the coherent artifact into account and yields evolution associated difference spectra (EADS) for a consecutive kinetic model and decay associated difference spectra (DADS) for a parallel kinetic model⁶⁷.