Ultrafast One- and Two-Dimensional UV Transient Absorption Spectroscopy of Haem Proteins

Proteins are dynamic macromolecules, which accomplish biological function driven by long-range interactions and global motions. While their biological function is often clear, little is known about the collective interactions which make these systems real molecular devices. The last decades showed an increased interest in accessing the very early events of protein function and the weak couplings among different sub-units. This implies the capability to observe events which occurs on sub-ps time scales. Ultrafast spectroscopy is a powerful tool to investigate in real time electronic processes and vibrational dynamics in photo-excited systems. The last years showed an increased interest in the UV spectral region, to access spectroscopic features of molecules with electronic excitations located exclusively below 400 nm. Among them, are the high- spin states of metal complexes and the aromatic aminoacids in proteins. Especially, exploring the UV range below 300 nm opened the possibility to employ naturally occurring aminoacids as local probes of protein dynamics and intra-proteins correlations. This allowed to access the dynamical evolution of sites which were previously spectroscopically silent, or whose spectra were too congested, in wild type proteins. In this thesis work, we focus on the study of Fe-based metal complexes, and in partic- ular on wild type haem-proteins in native environment, and we address the questions of the relaxation of the prosthetic group and its interaction with the Trp residues. Despite intensive study, the details of the early haem photocycle in haem proteins is still under debate. With this thesis work, we show the successful combination of our UV- extended broadband transient absorption setup with time gated fluorescence in unraveling the early mechanisms of haem relaxation. In particular, for the first time we found evidence that the electronic relaxation in ferric met-myoglobin is specific for the system and that the widespread analogy among the electronic relaxation in all myoglobin forms should be reconsidered. Despite their capability to access the time scales of Trp de-excitation, these techniques are not the most suitable to investigate the mutual interaction among several residues, nor can they access the dynamics of environmental fluctuations. Two-dimensional spec- troscopies are instead the right tool to solve these issues. To this purpose, we designed an ultra-broadband transient absorption two-dimensional setup in the UV, with a band- width exceeding 60 nm in excitation and 80 nm in detection. The performance of the setup, which is described in details, was successfully demonstrated on UV dyes and bio- logical samples and it represents an excellent complement to Fourier-transform 2D setups, whose biological application in the UV is often compromised by the limited observation bandwidth.