Search for: All records

Electron Paramagnetic Resonance (EPR) is an important technique for the investigation of the structure and function of metalloproteins and enzymes. The variety of questions in this line of research requires versatile instrumentation. In this work, we explored the utility of the open resonator concept for a general-use highly tunable TE011 resonator design at Q-band frequencies (≈ 34 GHz). Using proof-of-concept calculations, we establish a viable range of critical parameters compatible with the desired instrument specifications. We then present the resonator design, targeting ease of execution and handling. Experimental characterization of the built resonator shows high tunability. Specifically, we show that the resonator can be critically coupled and overcoupled with a three-fold change in the bandwidth using a matching short. We also show that the resonator can be incorporated with frequency tuning by means of movable axial plungers, allowing it to work with a wide range of samples using relatively narrow-bandwidth microwave instrumentation. Furthermore, because of its high tunability, the resonator is very tolerant of manufacturing imperfections, which makes it affordable and easy to execute with minimal tooling. We also discuss the long-term use of the resonator in our research, highlighting its versatility. 

The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H₂). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O₂sensitivity, binding specificity, or H₂production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O₂-tolerant [FeFe] hydrogenase fromClostridium beijerinckiiusing a flexible [GGS]₄linker group (CbHydA1-PsaE). We demonstrate that theCbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H₂at a rate of 84.9 ± 3.1 µmol H₂mg_chl^–1h^–1. Further, when prepared and illuminated in the presence of O₂, the nanoconstruct retains the ability to generate H₂, though at a diminished rate of 2.2 ± 0.5 µmol H₂mg_chl^–1h^–1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O₂-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H₂generating system that can function in the presence of O₂. 

[FeFe] hydrogenases comprise an important class of H2 evolving enzymes; however, these proteins are often oxygen sensitive and require anaerobic environments for characterization. Understanding the electrochemical relationships between various active and inactive states of these enzymes is instrumental in uncovering the reaction mechanisms of the complex six-iron active center of [FeFe] hydrogenases called H-cluster. Since states of the H-cluster exhibit distinct fingerprint-like spectra in the mid-IR range, IR spectroelectrochemical experiments provide a powerful methodological framework for this goal. This chapter describes protocols for performing Fourier-transform infrared (FTIR) spectroelectrochemical experiments on [FeFe] hydrogenases under anaerobic conditions. Topics included experimental design, data acquisition, and data analysis.