Session: Physical Chemistry Poster:

Introduction:
Nature’s inclusion of metals and metallo-cofactors in enzymes has led to the ability to perform very complex catalytic functions, such as, electron transfer/transport, nitrogen fixation, and biological hydrogen production, which are difficult to achieve with organic radical chemistry. Metallo-enzymes often employ first-row transition metals (Mn, Fe, Ni, Cu, V, etc.) in the active site. In order to fully understand the chemistry evolving within these enzymes, structural studies must be complemented with kinetic, spectroscopic, and computational studies.

Synchrotron-based X-ray crystallography diffraction provides high-resolution structural snapshots of metallo-enzymes at different equilibrium states and can be used to distinguish small changes of the atomic structure as an enzyme is stepped through the catalytic cycle. The catalytic cycle of redox enzymes often contains paramagnetic intermediates that can be studied using electron paramagnetic resonance (EPR) spectroscopy. In combination with X-ray crystallography, the magnetic-interaction tensors obtained from single-crystal EPR experiments can be directly related to the protein geometry. From such studies, a wealth of information on the catalytic mechanism of the enzyme can be obtained. However, many protein crystals used in X-ray crystallography are of dimensions in the 50-300 μm range and, as such, are too small to be studied using commercial EPR instrumentation.
Methods:
To study these limited sample volumes, new technical advances had to be developed. Here, we present a novel microwave resonant structure, the self-resonant micro-helix, for nanoliter samples that can be implemented in a commercial X-band (nominally, 9.5 GHz) EPR spectrometer.
Results:
The micro-helix was used in a first study with a 0.3×0.1×0.1 mm³ (3 nL) single crystal of [FeFe]-hydrogenase from Chlostridium pasteurianum (CpI) in the H_ox state and the full g-tensor within the molecular frame of the active site was determined. The g-tensor can then be used to validate quantum chemical calculations. Additionally, we show that advanced pulsed EPR spectroscopy could be collected from the same protein crystal, giving insight into the ¹⁴N hyperfine and quadrupole coupling of coordinated ligands.
Conclusions:
This work opens up, for the first time, the possibility of using advanced EPR techniques to study protein single crystals of dimensions typical for X-ray crystallography. Further applications of the new setup will be discussed.

Dr. Jason W. Sidabras

Assistant Professor, Medical College of Wisconsin

Appearances