Abstract
The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.
Highlights
Glutathione reductase (GR,1 EC 1.6.4.2, NADPH:oxidized glutathione oxidoreductase) is a pyridine nucleotide-disulfide oxidoreductase that shares many spectroscopic and structural properties with other members of this family such as lipoamide dehydrogenase, thioredoxin reductase, NADH peroxidase, and mercuric reductase (1)
The FAD-binding domain includes the FAD cofactor and a part of the GSSG-binding site, the redox-active disulfide bridge, which is located adjacent to the si-side of the isoalloxazine ring, and a His-Glu pair considered to be the acid-base catalyst in the catalytic electron transfer (3–5)
The reducing equivalents of NADPH are passed to GSSG via the isoalloxazine ring of FAD and the redox disulfide/dithiol center formed by two cysteinyl residues, Cys-58 and Cys-63 in the human enzyme (6)
Summary
Glutathione reductase (GR, EC 1.6.4.2, NADPH:oxidized glutathione oxidoreductase) is a pyridine nucleotide-disulfide oxidoreductase that shares many spectroscopic and structural properties with other members of this family such as lipoamide dehydrogenase, thioredoxin reductase, NADH peroxidase, and mercuric reductase (1). The reducing equivalents of NADPH are passed to GSSG via the isoalloxazine ring of FAD and the redox disulfide/dithiol center formed by two cysteinyl residues, Cys-58 and Cys-63 in the human enzyme (6). The first one represents a reductive reaction with the enzyme reduction by NADPH This step gives rise to the formation of a two-electron reduced species (EH2) with an oxidized FAD and a reduced dithiol center. One of the nascent thiols, i.e. the donor thiolate group of Cys-63, and the flavin form an intramolecular charge-transfer (CT) complex characterized by a broad absorption band peaking at 540 nm (1). In this step, the side chain of Tyr-197 fulfills a very important role. It has been postulated that this residue might serve as a gate in the NADPH-binding pocket, shielding the isoalloxazine ring of FAD (9)
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