The people involved: Katrin Adamczyk, Jens Dreyer, Erik T. J. Nibbering
Former team members: Oliver Dühr, Frank Tschirschwitz, Matteo Rini, Omar F. Mohammed, Karsten Heyne, Anwar Usman

International collaboration:
Ann-Kathrin Holm,^† Emad Mukhtar,^† Henk Fidder,^† Tomasz Zemojtel,* Thomas Dandekar,* Pavel M. Kozlowski,^ß Michael A. Cusanovich,º Jian Dong,^# Kyril M. Solntsev,^# Laren M. Tolbert^#
†: Department of Physical Chemistry, Uppsala University, Uppsala, Sweden
*: Department of Bioinformatics, University of Würzburg, Würzburg, Germany
*: European Molecular Biology Laboratory, Heidelberg, Germany
ß: Department of Chemistry, University of Louisville, Louisville, Kentucky, USA
º: Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona, USA
#: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA

Molecular function depends on the structure and relative geometry of the functional groups that make up the compounds. These compounds may function at the molecular level, but may also be implemented in supramolecular devices (nanotechnology). Structure-function relationships may in addition be understood by comparison of the characteristics of biomimetic compounds with biomolecular systems. Structure resolving techniques are thus essential tools in designing molecular compounds with optimal functionalities.

Light-triggered changes (i.e. photo-induced molecular transformations) enable an additional handle on the control of molecular functionalities, with the potential of exploring spatial and temporal domain parameters. Photo-induced transformations include trans/cis (E/Z) isomerization, ring opening and bond fission chemical reactions. The time-resolved elucidation of these chemical transformations from a structural point of view is thus of utmost importance, where the role of the excited state structure in the outcome of the reactions, the connection between reaction time scales and reaction efficiencies, and the important role of the surrounding medium have to be determined. Here we show some examples of the potential of femtosecond infrared spectroscopy to achieve these goals.

We have - until now - investigated the photophysics of photochromic switches (the spiropyran-merocyanine system) and of photosensor proteins (green fluorescent protein and photoactive yellow protein as well as relevant model chromophores).

Vibrational studies of the ring-opening reaction dynamics of spiropyrans:
ultrafast internal conversion and sequential product formation

1.  The molecule we investigated is 1',3',3',-trimethylspiro-[-2H-1-benzopyran-2,2'-indoline], commonly referred to as BIPS, and its 6-nitro-derivative (6-nitro-BIPS) dissolved in tetrachloroethene. The spiropyran compound can to first approximation be regarded as two nearly independent orthogonal halves: a chromene and an indoline. The BIPS molecule does not absorb in the visible. Upon absorption of a UV photon a ring opening and cis-trans isomerizations take place, resulting in a more or less planar molecule with a strong broad absorption around 540 nm. The huge shift in absorption wavelength can easily be understood from the fact that now the delocalized -system extends over the entire molecule. We have investigated with ~130 fs time resolution of one of the most studied photochromic reactions: the spiropyran-merocyanine chemical ring opening. The photochemical ring opening reaction is initiated by 70 fs UV pump pulses, and the time-evolution of the vibrational absorption spectrum toward the formation of merocyanine product species is directly followed by probing with ~100 fs tunable mid-IR pulses. Given the enormous research effort on these systems it is remarkable that up to now only a handful of studies have been performed with subnanosecond time-resolution.

2. Transient changes in the IR absorption spectrum of BIPS between 1430 and 1620 cm-1 at, from bottom to top, 2, 10, 20, 51, and 100 ps after UV excitation at 316 nm. For representation purposes the signals are displayed with the offset at consecutive delay times increased in steps of 0.8 mOD. The dashed lines indicate the zero signal level for the different traces. Lower panel: Steady-state IR spectrum of 50 mM BIPS (closed form) in tetrachloroethene.

3.  Comparison of the early and later transient response of 6-nitro-BIPS in tetrachloroethene and acetonitrile, showing a solvent dependent ground state recovery, and a solvent dependent merocyanine product formation.

4. Comparison between UV/vis and UV/IR transient data of 6-nitro-BIPS in tetrachloroethene. In this solvent a second isomer of merocyanine is formed sequentially with a 350 ps time connstant, from the first isomer that is generated with a 27 ps time constant.

5. The ultrafast internal conversion rates as function of the solvent-dependent energy gap of the first electronic transition of 6-nitro-BIPS enables a critical analysis of the energy gap law

NO-Myoglobin bond fission:
orientation of the nitric oxide ligand bound to heme iron

6.  The anisotropy of the ground state bleach signal of the NO stretching vibration, bound to the heme iron in myoglobin, reveals a configuration with NO tilted from the heme normal..

HBDI: Determination of the excited state structure of the chromophore of green fluorescent protein

7.  The anisotropy of the C=O stretching vibration of p-hydroxybenzylideneimidazolidinone (HBDI), the chromophore of green fluorescent protein, enables determination of the tilting of HBDI along the twisting coordinate upon electronic excitation.

PYP and model chromophores:
Determination of the excited state lifetime, quantum yield of ground state recivery and structure of the dark P and first intermediate I0 states of the chromophore of photoactive yellow protein

8.  Comparison of the anisotropy of transient absorption and bleach signals as obtained with polarization-sensitive IR spectroscopy enables the determination of the excited state lifetime of deprotonated trans-S-phenyl-thio-p-hydroxycinnamate, that appears to be strongly solvent dependent.