Multidimensional Nonlinear Femtosecond Spectroscopy of Biological Molecules

In this thesis photon echo spectroscopy is extended by measuring the spec- trum of the scattered signal (spectrally resolved photon echo) and by using two different wavelengths for the different applied laser pulses (two-colour photon echo), in order to investigate the ultrafast dynamics in biological molecules. To characterise the signal response of the spectrally resolved measurement a theoretical study using the Multimode Brownian Oscillator (MBO) model is performed. The MBO model describes the interaction of the analyte of interest and the surrounding environment in terms of a time- dependent correlation function M(t). The inverse linewidth of the measured signal spectra is found to correspond well with the temporal width of the photon echo pulse and an analytical fit to the signal is able to reasonably describe both the coupling strength and the dynamics of the time-dependent correlation function. The spectrally resolved and two-colour measurement techniques are ap- plied to a laser dye, rhodamine 101, to characterise the signal response (photophysics) of a molecule that does not undergo a chemical reaction. The measurement of the nonlinear signal spectra for rhodamine 101 proved in- valuable in identifying the different underlying photophysical processes that would otherwise have been difficult given only integrated intensity data. Another interesting result of measuring the signal spectra is the ability to characterise separately the ground and excited state dynamics. Information about the dynamics of fast processes that are otherwise difficult to deter- mine can be extracted using the two-colour technique. The combination of the spectrally resolved and two-colour techniques showed that AC-Stark effects are present and need to be considered when performing photon echo (transient grating) experiments. The new technique, two-colour spectrally resolved photon echo spec- troscopy, is applied to the biological system carbonmonoxy myoglobin (MbCO). Upon illumination MbCO undergoes a reaction where the carbonyl complex dissociates to form deoxy-myoglobin (deoxy-Mb). This study provides sup- porting evidence for the mechanism and the dynamics of the initial steps involved in this photodissociation reaction. We show that a change in spin state from the low spin bound state to the high spin unbound state occurs in the first 200 fs after excitation: the photophysics of photodissociation involve a second (intermediate) excited state with a lifetime of around 200 fs for MbCO and not vibrational cooling of a hot ground state as proposed in a previous model. Other photophysical time scales are also estimated: the Fe-CO bond breakage occurs within 25 fs and the lifetime of the Q-band levels is 35 fs.