Seeing Enzymes in Action with Laser T- jump Time-resolved XAS/XPE/XWAS Jung Y. Huang Keywords: liquid phase, metalloproteins, Laser T-jump, X-ray probe, pulse-to-pulse synchronization

Why study liquid-phase reactions? Majority of biological cellular processes and industrial applications occur in liquid phases. Water is a major contributor to a protein's 3-D structure and in reverse the protein also controls the structuring of its surrounding water.

Why metalloproteins?

It is estimated that about 1/4-1/3 of all proteins requires metals to carry out their functions. Metal ions involved are usually coordinated by nitrogen, oxygen or sulfur atoms belonging to amino acid residues of the protein. Metalloproteins play many different functional roles in cells, such as Storage: iron storage protein ferritin Transport: Oxygen transport proteins myoglobin and hemoglobin; Electron-transfer vectors for redox reaction such as Cytochromes (Fe), Plastocyanin (Cu), Chlorophyll-containing proteins (Mg) Enzymes: Hexokinase (Mg), methionine synthase (Co), Carbonic anhydrase (Zn), Superoxide dismutase (Cu), Nitrogenase (Mo) Signal Transduction: Calmodulin (Ca) Regulation: Transcription factors (Zn)

Dynamics in Biological Systems Protein structure and stability; folding/unfolding Protein Function Protein reaction kinetics Biological activity correlated with dynamic transition of structure (

Movements inside Proteins Many important biochemical processes occur on the time- scales of nanoseconds-microseconds.

Why Laser T-jump? The introduction of pulsed lasers excitation as triggers of the biochemical processes brought dramatic improvement in the experimental time resolution. However, this methodology is inapplicable to molecules without suitable chromophores. Laser T-jump methodology has evolved into one of the most versatile and generally applicable methods for studying fast biomolecular kinetics.

Why X-ray probes? Both e-beam and X-ray can give direct 3D structural information. However,  sca (hard X ray)=10 -3  sca (e). Electron beam cannot penetrate deeply into the bulk of a sample, thus it is limited to surface and gas-phase study. For condensed phase study, such as in liquid phase, several advantages can be yielded from X-ray probing technique, such as XAS, XPS, XRD, etc.

Why X-ray probes? The local structural methods are beginning to be applied to study excited-state structures of materials with the use of time- resolved pump-probe experiments.

Laser T-jump Time-resolved XAS/XPE/XWAS Target: Direct structural characterization of short-lived intermediates. Approach: Signal from delayed X-ray pulse probes the change in the electronic and spatial correlation function. Data Acquisition Procedure: Collect time-resolved X-ray scattering/absorption/emission data from -3  s to 3  s  q  S(q)/[EXAFS/XANES]  r  (r, t)/[  abs ( ,t)]  Spatial resolution 0.01A with  t=100 ps.

Pulse-to-Pulse Synchronization Timing Scheme Characteristics of the excitation laser: Pulse Repetition Rate (PRR): 347 kHz; 1/2 of the PRR of storage ring Pulse Energy: 4  J Excited size: 50x50  m 2

Pulse-to-Pulse Synchronization Timing Scheme

Further Consideration Estimated Signal Strength: For a dilute sample, signal from the excited solutes is about 0.01 of excited solvents. Assuming 10% (depending on  abs (  exe  and the focused laser intensity) optical excitation efficiency, S/BKG< How to improve the sensitivity? Use the chemical selectivity of XRA to distinguish the signal from excited solutes from the background signal.

Further Consideration

Non Pulse-to-Pulse Based Time-Resolving Technique

Conclusion To have a successful trXAS program for dynamic study of catalysts and proteins, we need a strong and coherent strategy for combining input from multiple experimental methods and theory (MD and models for structural retrieving). However, the reward can be high However, the reward can be high.