Electrostatic Control of the Isoalloxazine Environment in the Two-electron Reduced States of Yeast Glutathione Reductase

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.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). GR is a cytoplasmic flavoenzyme that is widely but not universally distributed in aerobic organisms. As shown in Equation 1, it catalyzes the reduction of GSSG by NADPH to form glutathione GSH.By maintaining an elevated [GSH]/[GSSG] ratio in most eukaryotic cells, GR participates in several vital functions such as the detoxification of reactive oxygen species as well as protein and DNA biosynthesis (1). Glutathione reductase has been isolated, characterized, and sequenced from different sources (1). The x-ray structures of the enzymes purified from human erythrocyte and Escherichia coli have been refined at high resolution (2-5). These structures show that GR is a homodimer. Each subunit (molecular mass ϭ 49 -59 kDa) contains one molecule of FAD and is composed of four domains, i.e. from the N-to the C-terminal end of the polypeptide chain, FAD-binding, NADPH-binding, central, and interface domains (3-5). The FAD-binding domain includes not only the FAD cofactor but also 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 (...

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