*4.4. Fluorescence Lifetimes*

Lifetimes were measured on an EasyLife V™ lifetime fluorometer (Madrid, Spain) with the stroboscopic technique, by using as excitation source a pulsed light of a diode LED operating at 278 nm. The number of channels used for each scan was 500, and the integration time was 1 s. Three scans were averaged in each experiment, and they were repeated twice at 25 ◦C in 50 mM phosphate buffer (pH 8.0) and 500 mM NaCl.

The experimental fluorescence decays (*D*(*t*)) were fitted to a sum of exponential functions: *D*(*t*) = *n* ∑ *i*=1 *ai* exp(−*t*/*τi*), where *τ*<sup>i</sup> is the the lifetime of the electronic excited states of the fluorescent species present in solution, and *ai* the pre-exponential factor of those electronic states. The pre-exponential factors can be interpreted not only in terms of the populations of the corresponding species, but also in terms of the radiative probability constants of Tyr residues, in the case of LrtA. We also determined the mean lifetime, <*τ*>, as: <sup>&</sup>lt; *<sup>τ</sup>* <sup>&</sup>gt;<sup>=</sup> *<sup>n</sup>* ∑ *i*=1 *fiτi*, where the *fi*s are defined as: *fi* = *aiτi*/∑*<sup>j</sup> ajτj*. The fitting procedure of the experimental fluorescence lifetime curves used an iterative method based on the Levenberg–Marquardt algorithm [51]. The temporal width of the excitation pulse, which distorted the observed decay, was taken into account through the instrument response function (IRF), which was determined by using a scattered solution of Ludox. Goodness of the fittings was tested by using a reduced χ2, that was calculated by measuring the spectral noise at time *t*, and determining the measurement uncertainties [52].
