Femtosecond pump-probe spectroscopy enables to follow in real time vibrational motions coupled to electronic transitions. Indeed, if the system is excited by a pulse shorter than the vibrational period, vibrational coherence is induced both in the ground and excited electronic states, providing information also on the excited state nuclear dynamics. In addition, time-domain vibrational spectroscopy circumvents the experimental difficulty of Raman spectroscopy in discriminating the low-frequency modes against the laser line.
In a pump-probe experiment, the output pulse train from an ultrafast laser, is divided into two beams: the sample is excited by one pulse train (pump) and the changes it induces in the sample are probed by the second pulse train (probe), which is suitably delayed with respect to the pump. Some properties related to the probe (reflectivity, absorption, luminescence, Raman scattering) is then monitored to investigate the changes produced by the pump in the sample.
The simplest of the pump-probe spectroscopy experiments is the measurement of the transmitted probe. In this case, one generally measures the change in the transmitted probe pulse energy induced by the pump as a function of the time delay between the pump and the probe pulses.
In collaboration with De Silvestri group in Milan, we have applied this technique to study the excited state dynamics of the blue copper protein azurin (Chem. Phys. Lett., 2002) and poplar plastocyanin (Biophys. Chem., 2003).
The figure below shows the time evolution of Azurin pump-probe signal resolved at a wavelength of 580 nm. The differential optical transmission decays exponentially with well visible superimposed oscillations.
The decaying component is due to the recovery of the ground-state population, while the oscillatory component can be ascribed to vibrational coherence created by the very short pump pulses in the excited and ground states.
By fitting the pump-probe signal decay with the sum of a decaying exponential and a constant offset, we found that the excited-state population returns to the ground-state within about 270 fs, in an essentially non-radiative fashion.
In order to analyse the oscillatory component
of pump-probe signal, the exponential fit was subtracted from the pump-probe
data and the residual shown in the left figure (low panel) was obtained.
The residual has been analysed in the frequency domain, by a Fourier Transform, to obtain the frequencies of the single vibrational components of the signal. The Fourier spectrum is shown in the figure below and it can be noted that all the vibrational modes obtained by conventional RR spectroscopy are retrieved. These are labelled with their frequency values in the figure:
Three low frequency modes can be observed between 30 and 80 cm-1. These bands have been associated to collective modes involving large biomolecule regions and of some biological relevance. It is interesting to note that the same modes have been also observed by inelastic neutron scattering spectroscopy in Azurin and Plastocyanin.
However, the most prominent feature in the Fourier spectrum of Azurin is the quite broad vibrational band at about 503 cm-1. Such a band is not present in Azurin RR spectrum and a damping time of about 300 fs, close to the value of the excited-state lifetime, has been estimated for the mode associated to this band. All these considerations could indicate that the 503-cm-1 band corresponds to an excited-state vibrational mode.
Poplar Plastocyanin shows a behaviour very similar to that of Azurin with the excited state lifetime of about 280 fs. The Fourier spectrum of oscillatory component of pPC pump-probe signal is shown in the left figure. Some of the major bands that appear in this figure are typical of the resonance Raman spectrum and, also in this case, an intense band is positioned at about 508 cm-1 whose dumping time is of about 300 fs.
Furthermore, in agreement with the previous results obtained for Azurin and with the neutron scattering data, three low frequency modes are visible between 20 and 80 cm-1.
As possible source of the 500-cm-1 vibrational mode has been proposed a Duschinsky rotation in the excited state. In order to obtain further insights relative to this mode, we have reproduced this vibrational band by a classical Molecular Dynamics (MD) simulation approach involving the excited state of poplar Plastocyanin copper site. The excited active site has been modellised by increasing the equilibrium bond length in the harmonic Cu-S(Cys84) interaction (Chem. Phys. Lett., 2001) and the power spectrum of the Cu-S(Cys84) bond distance fluctuation in the excited state is reported in the following figure. By increasing the force constant of the Cu-S bond from 110 to 290 kcal/(mol Ĺ), the band around 500 cm-1 could be reproduced (see figure below).
The fairly good agreement between the MD and the experimental results gives some support to the ‘hardening’ of the excited state modes as resulting from a Duschinsky rotation.