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 envir onment in electrochemical, photochemical, and chemical assemblies. Ele ctron-transfer communication between the protein redox site and its su rroundings is achieved either by covalent attachment of electron relay s to the protein or by using redox copolymers as electron mediators. G R is covalently attached to self-assembled monolayers of the N-hydroxy succinimide ester of cysteic acid formed by chemisorption of the respe ctive disulfide, 1, onto Au electrodes. The resulting GR monolayer ele ctrode is derivatized by N-methyl-N'-(carboxyalkyl)-4,4'-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 p otential, E = -0.72 V vs SCE on the electrode. The rate of GSH formati on is enhanced as the chain length linking the bipyridinium groups to the protein is increased. This enhancement in GSH formation is attribu ted to improved electrical communication with the enzyme active site. Photosensitized reduction of GSSG is achieved in a photosystem compose d of Ru(II) tris(bipyridine), Ru(bpy)(3)(2+), the protein glutathione reductase that is chemically derivatized by N,N'-bis(carboxyethyl)-4,4 '-bipyridinium (3), PAV(+)-GR, and EDTA as sacrificial electron donor. The formation of GSK in the photosystem is controlled by the electron -transfer quenching rate of the excited state. The electron relay unit s 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 copol ymer, 8, composed of N-methyl-N'-(3-acrylamidopropyl)-4,4'-bipyridiniu m (4) and acrylamide. The resulting protein-copolymer assembly affects the efficient photoinduced reduction of GSSG in the presence of Ru(bp y)(3)(2+) as photosensitizer and EDTA as sacrificial electron donor. I n this system, vectorial electron transfer from the excited state to t he protein redox site proceeds across the polymer backbone and the pro tein shell. Photosensitized reduction of GSSG by native GR has also be en accomplished by using N-methyl-N'(carboxyalkyl)-4,4'-bipyridimium p oly(L-lysine), PL-CnV2+ (9), as electron relay, Ru(bpy)(3)(2+) as phot osensitizer, 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 tha t the rate of electron transfer from the reduced,polymer, PL-CnV.+, to the enzyme redox site are controlled by the length of the tethers lin king the redox units to the polymer. With long chains, the electron me diator penetrates the protein backbone and attains short distances in respect to the protein redox center, resulting in enhanced electron tr ansfer. The rate constants for electron transfer From a series of redo x 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 sys tem, Pt catalyzes the reduction of protein-bound bipyridinium units by H-2. The reduced electron relay, PAV(.)-GR, mediates the electron tra nsport to the protein active center, where reduction of GSSG occurs.