Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

Abstract

Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultaneously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN^- ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH⁺, the super-reduced HsredH⁺ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates.

Fig. 1. Top, schematic representation of the H-cluster structure in the oxidised Hox state. Bottom, schematic representation of the principal H-cluster redox states and the electron and proton transfer events leading to corresponding state transitions.

Fig. 2. The spectroelectrochemical cell used in this study features a 3-electrode system with mirror-polished glassy carbon working electrode that simultaneously allows for electrochemical control of the protein crystal and detection by IR spectroscopy in reflection mode. (A) top view of the cell. (B) a representative CpI crystal as seen by visible microscopy, highlighting the area sampled by IR spectroscopy. (C) Schematic cross-section view of the cell (not to scale), including working electrode (W.E.), counter electrode (C.E.) and reference electrode (R.E.). The electrodes are embedded in a Delrin base plate.

Fig. 3. IR microspectroscopy of a single CpI crystal in its mother liquor. (A) spectrum of the untreated crystal with no applied potential (open circuit potential: approx. −200 mV). (B) Difference spectrum of the crystal after applying a potential of −367 mV (reduced minus oxidised). (C) Difference spectrum upon stepping back to −200 mV (re-oxidised minus reduced). Peaks marked by red wavenumber labels belong to the IR-signature of Hox.

Fig. 4. Comparison of redox titrations of CpI at pH 6, in crystallo (left column) and in solution (right column). The oxidised sample was reduced in steps from −200 mV to −583 mV. The sample was equilibrated for 15 minutes at each given potential. Each given potential is reported in the legend in mV. The top panels present baseline-corrected spectra obtained from (A) a CpI crystal or (B) a CpI solution. The inset shows a visible image of the crystal used. The middle panels present 2D plots of the titration for (C) the crystal or (D) the enzyme in solution. The bottom panels present titration plots for selected wavenumber positions representing each redox state for (E) the crystal or (F) the enzyme solution. For a detailed description of the peak assignments see the main text and refer to Table 1.

Fig. 5. Dissecting the protonated reduced states in CpI crystals. Zooming into specific spectral regions reveals marker peaks associated with single species. (A) from titrations at pH 6. (B) from titrations at pH 8. Colour legend is the same as Fig. 4A.