We aim to observe the structural changes that occur at the active site of enzymes during their catalytic cycle. We will construct a state-of-the-art 2D-IR (two-dimensional infrared) spectrometer and ultimately develop novel visible-pump 2D-IR-probe techniques capable of following reactions in real time. These methods will be used to study the hydrogenase family of enzymes. The objective is to determine the structure of the active site and transient changes of this structure during the reactive cycle. Two-dimensional infrared (2D-IR) spectroscopy is a new technique that completely transforms the amount of information available from the infrared spectrum of a given molecule. The 2D-IR method is directly analogous to multidimensional NMR spectroscopy, which has been invaluable in the field of structural biology but which is limited by millisecond time resolution. In contrast, 2D-IR is based on nonlinear femtosecond techniques that retain all the information regarding structural dynamics lost by NMR. 2D-IR also extends ordinary (1D) methods like FTIR through the presence of cross or off-diagonal peaks indicating coupling between vibrations. These cross peaks yield previously inaccessible information on molecular structure and vibrational dynamics.
"The majority of the original objectives of the proposal were achieved, to the extent that no serious modification was required over the course of the grant. The first ultrafast 2D-IR spectrometer in the UK was established and this has been extended to include transient 2D-IR and time-resolved infrared capability with time resolution from fs-ms. A significant number of results have been obtained relating to the solution phase structure, dynamics and photochemistry of the hydrogenase model compounds (8 publications total). Specifically, 2D-IR spectroscopy has been used to examine the solution phase structure, the vibrational relaxation dynamics and vibrational coupling of ligand groups surrounding the hydrogenase-like dimetal centre. 2D-IR lineshape analysis has examined the interactions between solvents of differing physical properties and the active site model while the reactivity in solution of a number of different model species has been investigated with time resolved IR methods. These have provided some important insights into the mechanism of reaction of these species. Finally, building on the latter photochemical studies, significant progress has also been made in using 2D-IR methods to follow reactions of these compounds in real time via transient 2D-IR methods (2 publications). This work has been sufficiently successful that it has been extended in a new direction towards encapsulation of model compounds in biomimetic gel matrices as preliminary nanomaterials for bioenergy applications (1 manuscript submitted).
One area requiring minor modification of the workplan was the work on enzyme active site dynamics. Unfortunately, the hydrogenases proved extremely troublesome in terms of their production and oxygen sensitivity during the long acquisition times needed for 2D-IR spectroscopy and so alternative systems were sought. This has lead to a research avenue studying iron-based NO sensing proteins and enzyme systems, using 2D-IR and time resolved IR to examine the role of dynamics and ultrafast fluctuations on biomolecule reactivity with regard to changes in ligand binding (2 publications to date plus 1 submitted and 1 in prep). While not the originally-envisaged system, these studies have successfully encapsulated the types of experiments described in the original workplan and have produced high quality outputs using 2D-IR to interrogate the role of ultrafast dynamics in biomolecule behaviour.
The results of the work and dissemination routes have led to a range of new funding opportunities and possible routes to future impact. In addition to exploitation of the knowledge gained from ultrafast spectroscopy of model hydrogenase systems for nanoscience and bioenergy applications mentioned above, the enzyme studies have laid the foundations for collaborative work with Diamond Light Source and the STFC Rutherford Appleton laboratory. Based at the Research Complex at Harwell, this project seeks to integrate 2D-IR with MX beamlines and theoretical modelling at the facility level and is partially supported by Strathclyde Knowledge Transfer funding."