Harnessing the Mechanism of Glutathione Reductase for Synthesis of Active Site Bound Metallic Nanoparticles and Electrical Connection to Electrodes

Abstract

It is demonstrated herein that the FAD-dependent enzyme glutathione reductase (GR) catalyzes the NADPH-dependent reduction of AuCl4-, forming gold nanoparticles at the active site that are tightly bound through the catalytic cysteines. The nanoparticles can be removed from the GR active site with thiol reagents such as 2-mercaptoethanol. The deep enzyme active site cavity stabilizes very small metallic clusters and prevents them from aggregating in the absence of capping ligands. The behavior of the GR-nanoparticle complexes in solution, and their electrochemical properties when immobilized on graphite paper electrodes are presented. It is shown that the borohydride ion, a known reducing agent for GR, is catalytically oxidized by larger GR-nanoparticle (>or=150 gold atoms) complexes generating catalytic currents, whereas NADPH (the natural reducing agent for GR) is not. It is proposed that the surface of the Toray graphite paper electrode employed here interferes with NADPH binding to the GR-nanoparticle complex. The catalytic currents with borohydride begin at the potential of GR-bound FAD, showing that there is essentially zero resistance to electron transfer (i.e., zero overpotential) from GR-bound FAD through the gold nanoparticle to the electrode.

Gold nanoparticle synthesis using the thermophilic bacterium Thermus scotoductus SA-01 and the purification and characterization of its unusual gold reducing protein

“…These types of proteins are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer by utilizing the energy obtained from the hydrolysis of ATP and have been found to be very diverse with respect to their physiological function and substrate unlike oxidoreductases which mainly catalyse oxidation-reduction reactions coupled to NADP/NAD + utilization [43][44][45]. Subsequent cloning and sequencing of the gene revealed the presence of a disulphide bond (between amino acid 337C and 481C) which confirms the hypothesis of Scott and coworkers [34], implicating an electron shuttle mechanism via a reduced disulphide bridge. This is further supported by the work done by Cason and co-workers [46] who found that reduction of the disulphide bonds resulted in electron transfer to a metal, in their case U(VI).…”

“…This was shown by Cason and co-workers [46] as well as Scott and co-workers [34]. Varying dithionite concentrations revealed that a stoichiometric ratio of less than 1:1 (4.6 μM sodium dithionite to protein) resulted in particles with defined edges.…”

“…Proteins present in cell-free extracts were routinely assayed for gold reducing ability by incubating the sample with 2 mM HAuCl 4 in 50 mM sodium phosphate buffer (pH 7.4) at 65°C for 24 h. Nanoparticle synthesis using the purified protein was performed under the same reaction conditions but with 4.6 μM sodium dithionite [33] added as a reducing agent [34]. In a living cell, natural reductants are present that are capable of reducing co-factors and in this case the disulphide bridge present in the protein, but these are absent in the purified protein fractions; thus, the reductant facilitates the reduced state of the protein.…”

Probing the nature of electron transfer in metalloproteins on graphene-family materials as nanobiocatalytic scaffold using electrochemistry

“…36 More recently, electrode surfaces prepared from nanomaterials facilitated protein ET have attracted particular interest due to excellent electrical conductivity, tunable surface chemistry and morphology. 37 Several groups reported that gold nanoparticles (AuNP) and carbon nanotubes (CNT) can serve as electron relays to electrically wire a range of redox proteins (e.g., cytochrome c (Cyt c), 38,39 glucose oxidase (GOD), 40 glutathione reductase, 41 and hydrogenase 23,42 to electrodes. Analogously, "molecular-wire"-like conjugated oligomers were also employed to wire the deeply buried redox site of a copper amine oxidase to a gold electrode.…”

The Metalloid Reductase of Pseudomonas Moravenis Stanleyae Conveys Nanoparticle Mediated Metalloid Tolerance

“…[16][17][18][19] GSHR is reported to reduce Au(III) to zerovalent form as well. 20 Thus, the class of pyridine nucleoside dependent oxidoreductases may represent an evolutionarily adaptable platform of inorganic ion reductases, with modifications to the enzyme altering metal ion selectivity. Such a catalytic center, with alterable precursor selectivity, is of interest in biogenic inorganic nanoparticle synthesis.…”