Electron-Transfer Communication in Glutathione Reductase Assemblies: Electrocatalytic, Photocatalytic, and Catalytic Systems for the Reduction of Oxidized Glutathione

Glutathione reductase, GR, is electrically communicated with its environment in electrochemical, photochemical, and chemical assemblies. Electron-transfer communication between the protein redox site and its surroundings is achieved either by covalent attachment of electron relays to the protein or by using redox copolymers as electron mediators. GR is covalently attached to self-assembled monolayers of the N-hydroxysuccinimide ester of cysteic acid formed by chemisorption of the respective disulfide, 1, onto Au electrodes. The resulting GR monolayer electrode is derivatized by N-methyl-Nʹ-(carboxyalkyl)-4, 4ʹ-bipyridinium (2) in the presence of urea. The relay-modified GR electrode exhibits electrical communication that leads to bioelectrocatalyzed reduction of oxidized glutathione, GSSG, to GSH upon application of a negative potential, E = -0.72 V vs SCE on the electrode. The rate of GSH formation is enhanced as the chain length linking the bipyridinium groups to the protein is increased. This enhancement in GSH formation is attributed to improved electrical communication with the enzyme active site. Photosensitized reduction of GSSG is achieved in a photosystem composed of Ru(II) tris(bipyridine), Ru(bpy)₃²⁺, the protein glutathione reductase that is chemically derivatized by N, Nʹ-bis(carboxyethyl)-4, 4ʹ-bipyridinium (3), PAV⁺-GR, and EDTA as sacrificial electron donor. The formation of GSH in the photosystem is controlled by the electron-transfer quenching rate of the excited state. The electron relay units linked to the protein act in the system as quenchers of the excited state and as electron mediators for electron transport to the protein active site. PAV⁺-GR was immobilized in the cross-linked redox copolymer, 8, composed of N-methyl-Nʹ-(3-acrylamidopropyl)-4, 4ʹ-bipyridinium (4) and acrylamide. The resulting protein-copolymer assembly affects the efficient photoinduced reduction of GSSG in the presence of Ru(bpy)₃²⁺ as photosensitizer and EDTA as sacrificial electron donor. In this system, vectorial electron transfer from the excited state to the protein redox site proceeds across the polymer backbone and the protein shell. Photosensitized reduction of GSSG by native GR has also been accomplished by using N-methyl-Nʹ-(carboxyalkyl)-4, 4ʹ-bipyridinium poly(L- lysine), PL-C_nV²⁺ (9), as electron relay, Ru(bpy)₃²⁺ as photosensitizer, and EDTA as electron donor. The rate of GSH formation is controlled by the tether length linking the redox units to the polymer backbone. Time-resolved laser flash photolysis experiments reveal that the rate of electron transfer from the reduced polymer, PL-C_nV^.+, to the enzyme redox site are controlled by the length of the tethers linking the redox units to the polymer. With long chains, the electron mediator penetrates the protein backbone and attains short distances in respect to the protein redox center, resulting in enhanced electron transfer. The rate constants for electron transfer from a series of redox polymers of varying spacer lengths to the protein redox center obey Marcus theory. Reduction of GSSG to GSH is also achieved by PAV⁺-GR using a Pt colloid and gaseous hydrogen as reducing agent. In this system, Pt catalyzes the reduction of protein-bound bipyridinium units by H₂. The reduced electron relay, PAV^.-GR, mediates the electron transport to the protein active center, where reduction of GSSG occurs.