Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution

Abstract

We report a versatile infrared spectroscopic method for studying redox chemistry of metalloproteins, and demonstrate for the first time electrochemically-induced changes to the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. A carbon particle network working electrode allows control over a wide potential window without the need for solution mediators.Hydrogenases are able to oxidise or produce H 2 at high turnover frequencies with a low overpotential requirement, and high selectivity for H 2 in the face of other gases. 1 A number of applications of hydrogenases have been explored, including light-driven H 2 production, 2 H 2 fuel cells, 3 and H 2 -supported recycling of the cofactor NADH. 4,5 Hydrogenases capable of functioning in the presence of O 2 are of particular interest for these applications. This impressive catalytic ability, together with the fact that hydrogenase active sites are built from readily available metals (iron, nickel), has inspired studies directed towards a detailed understanding of their chemistry. The presence of intrinsic CO and CN À ligands at the active sites means that infrared (IR) spectroscopy is a useful method for following electronic and coordination changes during catalysis or inhibition brought about by light triggers, gas exchange or electrochemical control. 6-9 Solution IR spectroelectrochemical studies interpreted alongside EPR analysis 10 and X-ray crystallographic structures have shaped our understanding of hydrogenase structure and mechanism, distinguishing a range of catalytically active and inactive states of the active site, with subtle variations evident between hydrogenases from different organisms. Most of the IR spectroelectrochemical studies on hydrogenases have been carried out in transmission geometry in an optically transparent thin layer electrochemical (OTTLE) cell originally developed by Moss et al. using a gold minigrid working electrode. 11 It is usually necessary to include small molecule redox mediators to achieve reasonable rates of electron transfer between the electrode and the hydrogenase; an exception is a study of the unusually small 49 kDa [FeFe]-hydrogenase from Chlamydomonas reinhardtii. 12 An Attenuated Total Reflectance (ATR) geometry opens up more flexible options for choice of working electrode. Rich and co-workers have developed an ATR-IR spectroelectrochemical cell in which a layer of protein coated onto the optical element is in contact with redox which shuttle electrons across the solution interface from a platinum mesh or glassy carbon electrode. 13 An alternative approach used widely for studying metallic surface chemistry, in which a thin metallic layer deposited on an ATR prism serves as the working electrode, has been adapted for biological molecules. 8,14 In this configuration, the protein is immobilised onto the gold electrode layer via a self-assembled alkanethiolate monolayer, and is in efficient solute contact, as demonstrated for reduction of hydrogenases upon introduction of H 2 . 8 Graphitic elect...

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

“…Despite the fact that solution assays on this enzyme demonstrated catalytic H 2 oxidation (albeit with a very low turnover frequency), the Ni a -R state(s) were not observed upon treatment with H 2 , 82,83,87 or electrochemical reduction in solution. 86 This lead to suggestion of a distinct two-state catalytic cycle for this enzyme involving only Ni a -SI and Ni a -C. 1,3 Recently, however, PFIRE spectra have revealed the presence of at least one Ni a -R state during catalytic H 2 oxidation, 40 suggesting that a common set of redox levels is conserved across the [NiFe] hydrogenases from quite different Groups.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…As a recent illustration, the [NiFe] regulatory Re Hase was entrapped in a carbon particle film deposited on a Si attenuated total reflectance prism. [243] The redox transitions between different states of the enzyme were determined with no need for redox mediators.…”

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

“…We have demonstrated previously that enzymes retain their native activity within an aqueous Nafion environment titrated to near-neutral pH. 10 Assays for proton reduction activity and N 2 reduction activity were conducted for both wild type and β-98 Tyr→His variant MoFe proteins in the presence and absence of Nafion as shown in Fig. S2 of the ESI †.…”

“…13 We chose to utilise carbon as the working electrode because this material exhibits minimal background proton reduction current down to potentials as low as –1 V vs. the standard hydrogen electrode (SHE). 10 The same cell design was used for both electrochemical and attenuated total reflectance (ATR)-IR spectroelectrochemical experiments (Fig. 1).…”