Nina Heidary

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.

Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase

Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.

Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells

Hydrogenases (H2 ases) are benchmark electrocatalysts for H2 production, both in biology and (photo)catalysis in vitro. We report the tailoring of a p-type Si photocathode for optimal loading and wiring of H2 ase through the introduction of a hierarchical inverse opal (IO) TiO2 interlayer. This proton-reducing Si|IO-TiO2 |H2 ase photocathode is capable of driving overall water splitting in combination with a photoanode. We demonstrate unassisted (bias-free) water splitting by wiring Si|IO-TiO2 |H2 ase to a modified BiVO4 photoanode in a photoelectrochemical (PEC) cell during several hours of irradiation. Connecting the Si|IO-TiO2 |H2 ase to a photosystem II (PSII) photoanode provides proof of concept for an engineered Z-scheme that replaces the non-complementary, natural light absorber photosystem I with a complementary abiotic silicon photocathode.

Raw data supporting article: Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells

Raw Data/Measurement results supporting article: Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells