Sample Preparation. Slr1694-DNA encoding the full-length SLR protein (amino acids 1–160; GenBank accession no. D90913, NP_441709), was amplified by PCR from Synechocystis
sp. PCC6803 DNA (kindly provided by I. Maldener, University of
Regensburg, Regensburg, Germany) and cloned between EcoRI and HindIII
sites of the pet28a(+) vector (Invitrogen). Protein was expressed in Escherichia coli
strain BL21(DE3) at 18°C in 0.7 mM IPTG overnight. Slr1964 was purified
on Ni-nitrilotriacetic acid resin (Qiagen, Hilden, Germany) according
to the supplier’s instruction. The eluate was dialyzed 2× again 200
volumes of 10 mM NaPi/10 mM NaCl (pH 8.0) and concentrated by
ultrafiltration (PM30; Millipore). For isotopic replacement, the
protein was concentrated and subsequently diluted with buffered D2O at least three times.
Femtosecond transient absorption spectroscopy was carried out with a
Ti:sapphire-based regenerative amplification system as described (38).
A 400-nm pump beam was obtained by frequency-doubling the output from
the amplifier and attenuated to 300 nJ. Femtosecond time delays up to 5
ns between pump and probe were controlled by a delay line, and
time-gated spectra at 109 delay times were recorded. The polarizations
of pump and probe beams were set at the magic angle (54.7°). The
instrument response function was fit to a Gaussian of 120 fs full-width
at half-maximum (FWHM). The samples were loaded in a flow system of
4-ml volume, including a flow cuvette of 1-mm path length, and flowed
by a peristaltic pump.
The transient absorption spectra were globally analyzed in terms of a
kinetic model with sequentially interconverting EADS, i.e., 1 → 2 → 3 →
…. The arrows indicate successive monoexponential decays with
increasing time constants, which can be regarded as the lifetime of
each species. The first EADS corresponds to the time 0 difference
spectrum. The EADS are not necessarily associated with pure molecular
species and often reflect mixtures of molecular species. To further
unravel the pathways for product-state formation, a target analysis was
applied, in which a specific kinetic scheme was tested (see Supporting Text
and Figs. 6 and 7). With this procedure, the SADS of the pure molecular
states are estimated. The global and target analysis procedures have
been extensively reviewed in ref. 39.