Infrared spectroscopy of the nitrogenase MoFe protein under electrochemical control: potential-triggered CO binding.

We demonstrate electrochemical control of the nitrogenase MoFe protein, in the absence of Fe protein or ATP, using europium(iii/ii) polyaminocarboxylate complexes as electron transfer mediators. This allows the potential dependence of proton reduction and inhibitor (CO) binding to the active site FeMo-cofactor to be established. Reduction of protons to H2 is catalyzed by the wild type MoFe protein and ß-98Tyr→His and ß-99Phe→His variants of the MoFe protein at potentials more negative than -800 mV (vs. SHE), with greater electrocatalytic proton reduction rates observed for the variants compared to the wild type protein. Electrocatalytic proton reduction is strongly attenuated by carbon monoxide (CO), and the potential-dependence of CO binding to the FeMo-cofactor is determined by in situ infrared (IR) spectroelectrochemistry. The vibrational wavenumbers for CO coordinated to the FeMo-cofactor are consistent with earlier IR studies on the MoFe protein with Fe protein/ATP as reductant showing that electrochemically generated states of the protein are closely related to states generated with the native Fe protein as electron donor.

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

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.