The completion of the self-resonant micro-helix (Fig. 02) opens up the possibility to study single-crystals with paramagnetic intermediates on crystals of dimensions less than 0.3 x 0.3 x 0.3 mm^3. These dimensions allow for studying protein crystals at the same volumes needed for structure determining x-ray crystallography diffraction experiments. The combination of structure and function determination is highly informative and highly sought after. Due to the very high efficiency parameter, the micro-helix geometry is advantageous in extending pulse EPR to experiments that usually require costly high-powered microwave amplifiers, further expanding the applicability of pulse EPR.
Although we have applied the micro-helix to study hydrogenase, the instrument is not limited to proteins. In fact, there are many fields of research, such as, molecular magnets, quantum computing, and material science which can benefit from the micro-helix geometry and EPR in general. To further the usefulness of the micro-helix for quantum computing, a micro-helix has been constructed of super conducting NbTi wire. The resonator is in the early stages of development but the increase in magnetic field will allow new quantum computing experiments.
This project maintains the long-term goal of studying enzymes structure and function which could not be previously studied due to lack of sensitivity. The self-resonant micro-helix has enabled, for the first time, the collection of EPR data from a 0.3 x 0.1 x 0.1 mm3 (3 nL) single crystal of [FeFe]-hydrogenase from Clostridium pasteurianum (CpI; [6Fe]-cluster) in the Hox state and the determination of the g-tensor (Fig01). Additionally, advanced pulse methods that measure the hyperfine coupling could be collected from the same protein single-crystal. The determination of the g-tensor and the ability to perform hyperfine experiments has an impact in both analytical and bioinorganic chemistry. Fundamentally understanding such enzymes is of broad biochemical and biophysical importance as we move towards bioengineering mimics of nature’s most elusive chemistry.
Currently crystals of suitable size (between 3-5 nL) are available of the [FeFe]-hydrogenase CpI in the Hox state and a reduced CpI-apo which has an EPR signal derived from the reduced four iron-sulfur clusters. In CpI-apo, the active center (H-cluster) is not present. The CPI-apo crystal could be used to study the electron transfer pathway of the [FeFe]-hydrogenase and how it relates to the function of the hydrogenase. It is also possible to obtain crystals in the inactive Hox-CO state, which may lend insight into reducing the oxygen sensitivity of the [FeFe]-hydrogenase. These samples will be studied in detail.
It is wholly expected to continue this line of research to maximize the socio-economic impact of this basic science research. Specifically in order to study the key roadblocks to creating the hydrogen economy using hydrogenase or similar enzymes. Some roadblocks include oxygen tolerance, miniaturisation of the protein backbone, and overall turn over rate.
