Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal

Abstract

We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.

Scheme 1. Active site redox levels of Hyd1 relevant to this study. Catalytically active states are labelled ‘Ni_a–X’, where X = SI, C, R or L.

Fig. 1. Schematic representation of the infrared microspectroscopic-electrochemical cell showing the placement of the crystal sample directly onto a glassy carbon working electrode (WE), and positioning of the reference and counter electrodes (RE, CE). Approximately to scale, as indicated.

Fig. 2. (A) Visible images, at 4× (left) and 36× (right) magnification, of a Hyd1 crystal deposited on the glassy carbon working electrode of the infrared microspectroscopic-electrochemical cell. A 15 × 15 μm² sampling area, used to record the IR spectra presented in (B), is indicated with black squares. Images were recorded using the visible setting on a Bruker Hyperion 3000 microscope. In addition to the Hyd1 crystal these images show fine scratches that remain in the glassy carbon surface following the polishing procedure. (B) (i) Reduced minus oxidised difference spectrum following a step from +241 to –359 mV, and (ii) re-oxidised minus reduced spectrum following a step from –359 to +241 mV. Dashed lines show the wavenumber positions of the intrinsic ν_CO bands of the Ni–B (blue), Ni_a–R_II and Ni_a–R_III (red) states of the Hyd1 active site.

Fig. 3. Infrared spectra recorded as a function of time highlight time-dependent formation of two Ni_a–R states, Ni_a–R_II and Ni_a–R_III. Spectra are presented as reduced minus oxidised baseline-corrected difference spectra following application of a reducing potential step from +241 to –359 mV and were recorded on the same Hyd1 crystal, and at the same position, as indicated in Fig. 2A.