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

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 N10–C10a 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 N1 and/or N5 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 N5 site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N5(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.

The oxidation-reduction potential of the E ox /EH 2 couple is Ϫ230 -250 mV at pH 7.0 and 20°C (8). 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 lies in the NADPH-binding pocket but moves aside when NADPH is bound. This movement allows the nicotinamide ring to come close to the isoalloxazine ring of FAD for electron transfer (2). It has been postulated that this residue might serve as a gate in the NADPH-binding pocket, shielding the isoalloxazine ring of FAD (9). The second half-reaction is oxidative. EH 2 reacts with GSSG yielding two molecules of GSH and regenerating the oxidized enzyme with its active disulfide site.
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In this reaction, many intermediates are formed. A stable mixed disulfide (MDS) 2 intermediate was proposed according the following equations (7).
MDS N E ox -GSH N E ox ϩ GSH (Eq. 5) In this catalytic scheme, the Cys-58 residue (human GR) is the interchange (or distal) thiol and interacts with GSSG (1,7). The acid-base catalyst of the interchange, i.e. the histidyl side chain of His-467Ј paired with a glutamate residue (Glu-472Ј), would have two roles; its primary function would be to protonate (acid catalyst) the first departing molecule of GSH, inhibiting the back reaction, i.e. attack of glutathione thiolate on the MDS (10). The second function (base catalyst) would be to stabilize the charge-transfer complex between FAD and the thiolate group of Cys-63 (6). Therefore, the interchange thiol, the charge-transfer thiol, and the His-Glu acid-base catalyst are closely linked structural elements in human GR. They are conserved in the E. coli and yeast sequences (1,11,12). A full understanding of the molecular mechanism of electron transfer in GR requires detailed information concerning the structures and environments of the redox sites. To better understand the role of the FAD cofactor in GR, we have undertaken a resonance Raman (RR) study on the two-electron reduced state, this redox state appearing to be of key importance in the function and regulation of the enzyme (4,6,7). RR spectroscopy has proven to be a valuable method for the investigation of the structure and the environment of the flavins in flavoproteins (13,14).
In this paper, we describe and analyze the RR spectra of the E ox , EH 2 , and MDS forms of the yeast GR. The two-electron reduced states were formed under controlled conditions using different reducing agents: NADPH, GSH, or dithiothreitol (DTT), in the absence or presence of GSSG or NADP ϩ . Although the three-dimensional structure of oxidized GR is known, and models of the reductive and oxidative half-reactions are available, many questions still remain unanswered. One of these is the importance of FAD in the GR catalysis. Our objective is to determine the possible influences of the substrates and products on the structure and environment of the isoalloxazine ring of FAD and to relate our observations to the function and regulation of the GR activity.

EXPERIMENTAL PROCEDURES
Materials-Yeast glutathione reductase was purchased from Sigma. The enzyme in an ammonium sulfate solution was dialyzed for 6 h in 50 mM potassium phosphate buffer, 0.5 mM EDTA, pH 7.2. To improve the protein purity, the solution was then applied onto a column of 2Ј,5Ј-ADP-Sepharose (Amersham Biosciences) (15). The elution of GR was done with a linear NaCl gradient (0 -1 M) added to the buffer (8). The pooled fractions were washed by repeated concentrations and dilutions with 10 mM potassium phosphate buffer, 0.5 mM EDTA, pH 7.2, on Centricon 50. The concentration of the eluted oxidized enzyme was spectrophotometrically determined using an extinction coefficient of 11,300 M Ϫ1 cm Ϫ1 at 462 nm (16). The measured activities of the purified enzyme were higher than 0.33 mmol of NADPH/min/mg of protein. The NADPH concentration was calculated using ⑀ 340 ϭ 6,220 M Ϫ1 cm Ϫ1 (17). NADPH, GSSG, GSH, and DTT were purchased from Sigma. All other chemicals were of the highest grade commercially available.
The two-electron reduced forms of the enzyme, EH 2 and MDS, were generated by reduction of oxidized GR (0.6 -0.8 mM for the Raman experiments) with NADPH, GSH, or DTT in the presence or absence of GSSG or NADP ϩ . These reductions were performed under anaerobic conditions in equilibrating independently the enzyme and the solutions of reactant with wet argon (18). After equilibration, a small aliquot of a solution of NADPH or GSH was injected through a septum in a cuvette containing the solution of GR. The color change of the enzyme from yellow to orange-red as well as the concomitant changes in absorption spectrum proved the formation of the EH 2 or MDS species. To maintain the enzyme in the two-electron reduced state, the spectroscopic measurements were performed under anaerobic conditions using a continuous flow of wet argon over the surface of the sample. Under these conditions, the EH 2 and MDS samples were stable for several hours. The reduction of GR with DTT was conducted by dialyzing the protein at 4°C in a 50 mM potassium phosphate buffer, pH 7.2, containing 0.5 mM DTT. This reduction produces an EH 2 species that is stable for several hours, even in the presence of air.
Using electronic absorption and RR spectroscopies, the enzyme stability under laser irradiation was checked. No spectral variation was observed during the experiments. After the RR experiments were done, we controlled the reversibility of the enzyme reduction in regenerating the fully oxidized form by dialysis and in measuring the enzyme activity. Such controls showed that the GR activity was stable during the spectral determinations.
Spectroscopy-Resonance Raman experiments were recorded at 20 Ϯ 1°C on a Jobin-Yvon spectrometer (Ramanor HG2S-UV) using argon (Coherent, model Innova 100) and helium/cadmium (Liconix, model 4050) lasers. The signal-to-noise ratios were improved by summation of individual spectra. Spectral treatments (addition, subtraction, and removal of broad and featureless fluorescence backgrounds) were made using the Grams 32 software (Galactic Industries) (19). The frequency precision was 0.5-1 cm Ϫ1 for the most intense RR bands and 1.5-2 cm Ϫ1 for the weakest bands. Fig. 1a shows the high frequency region of the RR spectrum of oxidized GR, excited at 441.6 nm. In this study, the band numbering previously adopted for the prominent RR bands of oxidized flavoenzymes has been used (20). In the 1300 -1700 cm Ϫ1 region of the spectra, four bands observed at 1626 (band I), 1579 (band II), 1402 (band VI), and 1353 (band VII) cm Ϫ1 dominate. However, shoulders on the high frequency sides of band I (at 1643 cm Ϫ1 ), band III (at 1545 cm Ϫ1 ), band IV (at 1498 cm Ϫ1 ), and band V (at 1461 cm Ϫ1 ) are also identified. The 1200 -1300 cm Ϫ1 region of the RR spectrum shows a complex pattern with overlapping bands at 1224, 1248, 1261, and 1273 cm Ϫ1 (Fig. 2a). The frequency of the RR bands observed in the 1000 -1700 cm Ϫ1 region of oxidized GR (E ox ) are listed in Table I. The addition of either GSSG (up to 60 eq) or NADP ϩ (up to 100 eq) to oxidized GR has no influence on the RR spectrum (spectra not shown).

RR Spectra of Oxidized GR-
RR Spectra of NADPH-reduced GR-The RR spectra of GR anaerobically reduced with NADPH (2.5 eq) at pH 7.2 are FIG. 1. 1300 -1670 cm ؊1 regions of RR spectra of GR reduced by NADPH at pH 7.2. a, oxidized GR. b, GR reduced with 2.5 eq of NADPH. c, GR reduced with 2 eq of NADPH, in the presence of 100 eq NADP ϩ . Excitation was 441.6 nm. Summations were of 60 -90 scans. The asterisks above the three spectra indicate plasma lines. The band marked with L above spectrum c is due to the 1337 cm Ϫ1 band of free NADP ϩ . a.u., --. displayed in Figs. 1b and 2b. When compared with the RR spectra of the oxidized form ( Figs. 1a and 2a), those of the NADPH-reduced GR have lower signal-to-noise ratios. However, we clearly identify significant shifts for bands II (ϩ2 cm Ϫ1 ), V (ϩ3 cm Ϫ1 ), VI (ϩ5 cm Ϫ1 ), and VII (Ϫ2 cm Ϫ1 ). The 1200 -1300 cm Ϫ1 regions also exhibit substantial modifications. The band observed at 1248 cm Ϫ1 for oxidized GR is markedly diminished upon enzyme reduction, whereas we notice an increase in relative intensity and a broadening of the 1262 cm Ϫ1 band for the NADPH-reduced form (Fig. 2b). Band XI is apparently upshifted from 1224 cm Ϫ1 for oxidized GR to 1230 cm Ϫ1 for NADPH-reduced GR (Fig. 2, a and b).
When GR is reduced by NADPH (2 eq) at pH 7.2, the addition of NADP ϩ (15-150 eq) has no significant influence on the frequencies of the RR bands ( Fig. 1c and Table I). However, a slight broadening of these bands is detected.
RR Spectra of GSH-and DTT-reduced GR-The titration of E ox with GSH produces gradual upshifts of the RR bands II, III, V, VI, IX, and X (spectra not shown). With a saturating concentration of GSH (15-20 eq), the RR spectrum of the GSHreduced enzyme exhibits maximal shifts of ϩ3-6 cm Ϫ1 for bands II, III, V, and VI, when compared with the spectrum of oxidized GR (Fig. 3a versus Fig. 1a and Table I). As in the case of the NADPH-reduced enzyme, band X is apparently upshifted from 1248 to 1262 cm Ϫ1 (Fig. 2, a and c). The comparison of the 1300 -1670 cm Ϫ1 regions of the spectra of NADPH-reduced GR and GSH-reduced GR shows frequency shifts for bands II (from 1581 to 1584 cm Ϫ1 ), III (from 1546 to 1549 cm Ϫ1 ), and VII (from 1351 to 1354 cm Ϫ1 ) ( Fig. 1b versus Fig. 3a). In the 1200 -1300 cm Ϫ1 regions, the RR spectrum of the GSH-reduced enzyme closely resembles that of the NADPH-reduced form ( Fig. 2b and c and Table I). The RR spectrum of GR reduced with DTT at pH 7.2 is essentially identical to that of the enzyme reduced with GSH (Figs. 2a and 3b). The broad band XIV, however, appears to be downshifted (Table I).
Combined effects of GSH, GSSG, and pH produce different ratios of EH 2 and MDS (7). The RR spectrum of GR reduced with GSH at pH 8.4 is shown in Fig. 3c. The RR spectra of GR reduced with GSH (15 eq) at pH 6.3 in the presence of various concentrations of GSSG (2-60 eq) are not significantly different from each other (spectra not shown). The observed frequen-cies are listed in Table I, and one of these spectra is displayed in Fig. 3d. The comparison of the RR spectrum of GR reduced with GSH at pH 8.4 with that of GR reduced with GSH at pH 6.3 in the presence of GSSG shows a significant downshift and broadening of bands II (from 1585 to 1583 cm Ϫ1 ) and III (from 1551 to 1548 cm Ϫ1 ) (Fig. 3, c and d). In the lower frequency regions, bands X (1263/1261 cm Ϫ1 ), XI (1229/1227 cm Ϫ1 ), and XIV (1061/1058 cm Ϫ1 ) are also slightly affected (spectra not shown and Table I).
We have investigated the effect of NADP ϩ on the GSHreduced enzyme at pH 7.2. The RR spectrum obtained for GR reduced with GSH (15 eq) at pH 7.2 in the presence of NADP ϩ (15 eq) is very similar to the spectrum obtained for GR at the same pH in the absence of NADP ϩ . Small differences concern a slight broadening of most of the RR bands and the detection of a downshift of band II from 1584 cm Ϫ1 in the absence of NADP ϩ to 1583 cm Ϫ1 in its presence (spectra not shown and Table I).

RR Spectra of the E ox , EH 2 , and MDS Species of Yeast GR
Oxidized GR-The assignment of the RR bands of the isoalloxazine ring is based on isotopic data and normal mode vibrational analysis (19 -23). Fig. 4 shows the chemical structure and the numbering of the isoalloxazine ring, and Table I summarizes the band assignments for the oxidized and twoelectron reduced forms of yeast GR. The RR spectra of oxidized GR were previously investigated (24,25). Our study shows frequencies close to those published in the study of Schmidt et al. (24). With reference to the RR frequencies of FAD in water, Raman diagrams were previously drawn to visualize the apoprotein effect on the high frequency modes of the isoalloxazine ring (19,26). In the diagram of oxidized GR (Fig. 5), the frequency shifts detected for GR are generally negative, indicating an electron-deficient isoalloxazine ring (19,26). The shapes of the Raman diagrams were associated with the electron distribution through the three rings of the isoalloxazine moiety and correlated with the biological activities of several flavoproteins (19,26). The profile of GR is very different from those previously obtained for electron transferases or oxidases (19,26).
NADPH-reduced GR-The reduction of GR with NADPH provides an EH 2 complex (Equation 2)). The reaction product, NADP ϩ , forms a complex with EH 2 (K D ϭ 70 M), but NADPH in excess forms a tighter EH 2 -NADPH complex (K D ϭ 2.1 M) (27). This latter complex enhances the thiolate-flavin CT band (9).
The RR spectra of NADPH-reduced GR are in agreement with an oxidized isoalloxazine ring (Table I) (13,14,19,25). The comparison of the RR diagrams drawn for oxidized GR and its NADPH-reduced form illustrates the influence of the disulfide reduction on the isoalloxazine modes (Fig. 5). Upshifted frequencies are observed for most of the RR bands of the NADPH-reduced GR, indicating an increased electron density of the isoalloxazine ring upon dithiol formation. However, the isoalloxazine ring remains electron-deficient. This low electron density is likely at the origin of the stable formation of the CT complex between FAD and the proximal thiolate group constituting the dithiol active site (1). The changes in shape of the Raman diagrams also indicate a modification in the electron distribution through the isoalloxazine ring (Fig. 5).
For an enzyme preliminarily reduced with excess NADPH (EH 2 -NADPH), the displacement of the bound NADPH molecule by NADP ϩ has no influence on the RR modes of FAD (Table I). Although NADPH binding causes an important protein reorganization around Tyr-197 (2, 4), the structure and environment of the FAD do not therefore discriminate the presence of either NADPH or NADP ϩ in the NADPH-binding site of EH 2 .
GSH-and DTT-reduced GR-Glutathione is the physiological product of the GR catalysis. With a redox potential (E m7 ) of Ϫ234 mV, it is capable to reduce oxidized GR (1, 7, 16, 28). This reaction does not represent the reversal of Equation 3. It was suggested that the back reaction is blocked at the MDS stages (Equations 5 and 4) (7, 28). Direct evidence for formation of MDS was provided by x-ray crystallography using human GR crystals soaked with GSH (2, 4). The MDS-GSH species accumulates when GSSG is added and/or pH is decreased (7). The absorption spectrum of MDS appears to maintain an absorbance at 540 nm very similar to that of the CT complex (7,28).
DTT can reduce GR, considering the absorption spectrum of DTT-reduced GR that exhibits the 540 nm CT band (spectrum not shown). The formation of an EH 2 state by opening of the disulfide redox center of GR (E m7 ϭ Ϫ242 mV) is expected considering the redox potential of DTT (E m7 ϭ Ϫ327 mV) (1, 8,  29).   Table I, and those of FAD in water are from Desbois et al. (26).
The RR frequencies observed for GR reduced at pH 7.2 with either GSH or DTT again correspond to an oxidized flavin ring (Table I) (13,14,19). Taking into account the different uncertainties on these frequencies, the diagrams obtained for GR reduced with GSH or DTT are identical but differ from that obtained for NADPH-reduced GR (Fig. 5 and diagram not shown). On one hand, the increases in frequency of bands II and III observed upon NADPH reduction are lower than those detected using either GSH or DTT as a reductant. On the other hand, the frequencies of bands IX-XII are relatively less upshifted in the spectra of the GSH-or DTT-reduced enzyme (Fig.  5). For the GSH-or DTT-reduced forms, the most important frequency shifts concern bands II (ϩ5 cm Ϫ1 ), III (ϩ4 -7 cm Ϫ1 ), VI (ϩ5-6 cm Ϫ1 ), and X (ϩ14 cm Ϫ1 ) (Fig. 5 and Table I). Although the Raman diagrams of the GSH-or DTT-reduced GR show a gain in electron density, it is again worth noticing that the isoalloxazine ring remains electron-deficient in these twoelectron reduced states.
Effects of GSSG and of pH on the GSH-reduced Enzyme-When GR is reduced by GSH, the dithiol form of EH 2 and the MDS species are in equilibrium (Equations 4 and 5). In GR reduced with GSH at pH 8.4, the imidazole ring of His-467Ј partially deprotonates, likely limiting the formation of MDS (7). On the contrary, GR reduced with GSH at pH 6.3 in the presence of GSSG can accumulate the MDS species (7). An intermediate situation occurs for GR reduced by GSH at pH 7.2. Considering the RR spectra of GSH-reduced GR at pH 8.4 and 7.2, and in the presence of GSSG at pH 6.3, the expected increase in MDS concentration produces small downshifts of bands II (1585 to 1583 cm Ϫ1 ), III (1552 to 1548 cm Ϫ1 ), X (1263 to 1261 cm Ϫ1 ), and XIV (1061 to 1058 cm Ϫ1 ) ( Table I). These shifts indicate that the structure of the isoalloxazine ring and/or the environments of the N 3 H and either N 1 or N 5 sites are slightly affected by the thiol-disulfide interchange. In the crystallographic structure of human GR, the interchange thiol, the sulfur of Cys-58, is far from the flavin ring (3,4). Nevertheless, the binding state of this atom is likely controlled by Cys-63, proximal to the flavin, and His-467Ј. On one hand, the thiolate anion of Cys-63 interacts directly with the flavin ring (2-4). On the other hand, His-467Ј participates via its imidazole ring to the cleavage of the GSSG substrate and strongly interacts with the N 3 H group of FAD through its carbonyl peptide group (3). Therefore, the isoalloxazine ring of FAD is sensitive to the formation of MDS through slight changes in its electrostatic environment.
Bands II and III-The main differences in RR spectra of the EH 2 and MDS species concern the frequencies of bands II and III (Table I). These bands were associated with the N 1 ϭC 10a -C 4a ϭN 5 region, especially stretching modes of the N 1 -C 10a and C 4a -N 5 bonds (Fig. 4). The characterization of these modes has a particular importance considering the involvement of the ring II-ring III junction in the chemical reactivities as well as the redox properties of the flavins (30,31). On one hand, the band II frequency was found sensitive to out-of-plane distortions of the isoalloxazine ring (19). On the other hand, the frequencies of bands II and III were found affected by hydrogen bonding at the N 1 /N 5 sites (19). In this latter case, a linear relationship was determined between the frequencies of bands II and III of planar isoalloxazine ring systems. In fact, the frequency of the (C 4a -N 5 ) (or (C 10a -N 1 )) mode is decreased when the strength of the C 4a -N 5 (or C 10a -N 1 ) bond is decreased as a result of hydrogen bonding at N 5 (or N 1 ) (19). The frequencies of bands II and III of GSH-reduced GR (1584 and 1549 cm Ϫ1 , respectively) follow the linear relationship previously determined for nearly planar isoalloxazine rings (see Fig. 8 in Ref. 19). Those of the NADPH-reduced enzyme (1581 and 1546 cm Ϫ1 , respec-tively) show a small deviation from this linear correlation. All of these observations indicate that the isoalloxazine conformation is slightly distorted in the EH 2 state generated by NADPH and practically planar in the enzyme reduced by GSH. They also indicate variations in hydrogen bonding at the N 1 and/or N 5 site(s) (19)

(see below).
Band VII-For GR reduced with NADPH, RR band VII is detected at 1351 cm Ϫ1 . In the spectra of GR reduced with GSH or DTT, the corresponding band is shifted to 1354 -1355 cm Ϫ1 (Table I). Band VII was assigned to a ring II mode, with a major contribution of N 10 -C 10a stretching (20 -22) (Fig. 4). The band VII upshift can be attributed to a change in electronic or steric interaction between the hydroxyl group of Tyr-197 (human GR) and the N 10 -C 10a portion of the FAD. In the GSH-reduced enzyme, this hydroxyl group is in close contact with the N 10 (FAD) atom (3). Upon NADH binding, the aromatic side chain moves away so that the nicotinamide ring can reach the flavin (3). Therefore, the frequencies of bands II, III, and VII appear to distinguish the EH 2 state generated with NADPH from that generated with GSH or DTT.
Band X-Band X of oxidized GR was assigned at 1246 cm Ϫ1 on the basis of its large sensitivity on N 3 H deuteration (19). This band corresponds to a stretching mode of the N 3 -C 4 and C 2 -N 3 bonds and is expected to be sensitive to the hydrogen bonding state of the N 3 H site (19 -22) (Fig. 4). In oxidized GR, the N 3 H group of the isoalloxazine ring of one subunit is tightly bound to the carbonyl group of His-467Ј of the other subunit (3,5). The apparent upshift of band X from 1248 cm Ϫ1 for oxidized GR to 1261-1263 cm Ϫ1 for its reduced forms indicates an increased interaction of the N 3 H(FAD) group with the protein and/or the solvent. This effect is likely linked to the 0.25 Å shift of the flavin ring and/or the slight increased bending of the isoalloxazine ring, observed upon E ox /EH 2 transition (4). Our RR data clearly indicate that the N 3 H-protein interaction is stronger in the EH 2 and MDS states than in E ox .

Structure and Environment of the Isoalloxazine Ring in the EH 2 and MDS States
The two-electron reduction of GR with either NADPH or GSH leads to EH 2 and/or MDS species, stable intermediates that are extremely important in the catalysis (1,6,7). An opening of the disulfide redox center occurs in both the EH 2 and MDS states. The difference between these forms concern the binding state of the interchange thiol that is either free in the EH 2 states or engaged in a disulfide bond with GSH in the MDS states (4).
The RR frequencies observed for NADPH-and GSH-reduced GR indicate the existence of two different EH 2 forms. In particular, the different frequencies of bands II and III indicate that the ring II-ring III junction has a different structure and/or environment (Table I). In this line, the edge formed by the N 5 , O 4 , and N 3 H atoms of FAD as well as the si-side of FAD are surrounded by a number of polar or charged groups in interaction with water molecules (3)(4)(5). For human GR, the thiol or thiolate groups of the redox center (Cys-58 and Cys-63) and the Lys-66/Glu-201 and His-467Ј/Glu-472Ј pairs were associated with the catalytic activity of GR (1). All of these residues are conserved in the amino acid sequence of yeast GR (12). To explain the differences in frequency of bands II and III, several possibilities are envisaged as follows.
FAD-Thiolate Interactions-A CT complex between the C 4a flavin atom and the S(thiolate) donor group of the disulfide/ dithiol center has been postulated for the EH 2 and MDS states (4). The RR diagrams show an important deficit in electron density for the isoalloxazine ring of oxidized GR. The positive shift of bands II and III upon NADPH, GSH, or DTT reduction is therefore a consequence of a stable electronic interaction between the negative S(thiolate) donor and the C 4a -C 10a junction.
A first hypothesis is to consider that the S(thiolate)-FAD interaction differs in the NADPH-and GSH-reduced forms of GR, provoking different electron densities at the C 4a and/or C 10a atom(s) of FAD. The absorption spectra of the NADPH-, GSH-, and DTT-reduced forms of GR are very similar. In particular, we notice the presence of a CT band at 540 nm (Ref. 16 and this work). An intensification of the CT band was previously detected for the EH 2 -NADPH complex (9). This hyperchromic effect likely corresponds to the parallel positioning of three rings in this complex, i.e. the flavin, nicotinamide, and Tyr rings (2). The MDS species has an extinction coefficient at 540 nm almost as high as the thiolate-flavin CT complex of EH 2 (7,28).
The maintenance of a 540 nm band for the EH 2 and MDS species of GR suggests no change in the CT interaction between FAD and its thiolate donor. A modification in protein conformation is expected to affect the strength of the CT complex and thus the position of the CT band. This effect is clearly observed when the absorption spectra of the EH 2 forms of various pyridine nucleotide-disulfide oxidoreductases are compared. With different protein environments around the FAD and/or the disulfide/dithiol center, the CT band position varies between 500 and 580 nm (32)(33)(34)(35)(36).
The x-ray structure of NADH-reduced GR indicates that the CT complex between the S(thiolate) of Cys-63 and the C 4a (FAD) atom is stabilized by the dipole of a well oriented ␣-helix as well as by two hydrogen bonds with the hydroxyl groups of Thr-339 and of the ribityl chain (O 2 Ј(FAD)) (4, 5). The imidazolium group of His-467Ј was also proposed to be involved in the transfer of reducing equivalents from the reduced flavin to the disulfide center (4). The crystal structure of the GSHreduced enzyme gives no indication of a significant change in the distances between the S(thiolate) atom and its plausible partners (4). Therefore, the different RR spectra observed for the EH 2 and MDS states of GR may be interpreted in the frame of a modified intramolecular CT complex between FAD and S(thiolate). It is important to note that the available absorption and crystallographic data are hardly compatible with this interpretation.
Hydrogen Bonding States of the N 5 (FAD) Site-Except for proton movements, the x-ray structures of GR yield a geometric picture of the catalytic cycle (2-5). Electrons flow from NADPH to GSSG via the isoalloxazine ring and the redox active disulfide without a major conformational change of the protein. The GSH-and NADPH-binding pockets are physically well separated in the GR active site, with the FAD and the redox-active disulfide bridge lying in between (2). Considering this structural arrangement, the mechanisms by which the EH 2 and MDS species are formed differ. The E ox reduction with NADPH is mediated by FAD, which is transiently reduced (6,37). In this case, the electron and proton transfers are oriented from the re-side to the si-side of the flavin (4). On the contrary, the GR reduction by GSH likely proceeds via the si-side by direct opening of the disulfide center to form a dithiol site and subsequently MDS (7). The different frequencies of bands II and III indicate various degrees of hydrogen bonding at N 1 and/or N 5 for the MDS form as well as for the two EH 2 forms (19). Therefore, in influencing the electrostatic environment of the N 1 /N 5 atom(s) of FAD, GR would discriminate between the two pathways by which the disulfide center was reduced.
The crystal structures of GR show that the isoalloxazine ring of FAD is buried in a deep pocket of the protein, out of the direct influence of solvent molecules (2, 5). The N 1 atom has practi-cally no interaction with the protein or the solvent. A distance of 3.49 Å between N 1 (FAD) and the NH(peptide) group of a conserved Thr residue only suggests the formation of a very weak hydrogen bond (4,5). On the contrary, the environment of the N 5 (FAD) atom is relatively polar. The ammonium group (NZ) of a conserved lysine residue (Lys-66 and Lys-50 in human and E. coli GR, respectively) is at a distance of 2.96 -3.01 Å (4,5). Moreover, a network of hydrogen bonds is probably organized around this NZ(Lys) atom. For oxidized GR and GSHreduced GR, the carboxylate group of a paired glutamate residue (Glu-201 and Glu-181 in human and E. coli GR, respectively) forms a salt bridge with NZ that also interacts with two water molecules (numbered 500 and 568 in human GR) and the O 4 (FAD) atom (4,5). In the GSH-reduced enzyme, NZ(Lys-66) is consequently within hydrogen bond distances with N 5 (FAD) (3. (2.76 Å). Taking into account these structural data, the nature as well as the strength of the interaction between NZ(Lys) and N 5 (FAD) are not clear because with a maximum of three protons for the NH 3 (Lys-66) group, all of the potential hydrogen bonds indicated in the crystal structures cannot be engaged simultaneously. From the different frequencies of bands II and III, it seems possible to relate the different hydrogen bonding states of N 5 to different configurations of the terminal NH 3 (Lys-66) group in the NADPH-and GSH-reduced enzymes. Our RR data are consistent with an increased hydrogen bonding state of the N 5 (FAD) atom in passing from NADPH-reduced GR to GSH-reduced GR. A discrete rotation of the ammonium protons could be at the origin of an hydrogen bonding rearrangement around N 5 in the EH 2 and MDS species. A more diffuse mechanism could, however, be envisaged. In GR reduced by NADPH, the transient formation of reduced FAD induces the N 5 protonation (6,37). The interaction between NZ(Lys-66) and N 5 is therefore expected to be strongly modified. Upon reoxidation of FAD, a positive density of charge can be moved from N 5 to the NH 3 (Lys-66) group and then to more distant proton acceptors/donors in the protein. In the absence of transhydrogenase activity, the GR reduction by GSH could result in a different network of hydrogen bonds around NZ and N 5 .
Finally, it is interesting to note that a conserved Lys-Glu pair occupies an homologous position in the structures of lipoamide dehydrogenase, the ammonium function of Lys being near the O 4 and N 5 sites of FAD (38,39). The mutation of the conserved Lys-35 by an Arg residue in E. coli lipoamide dehydrogenase influences the enzyme properties (40). In particular, the redox potential of FAD was raised by ϩ60 mV. In contrast, that of the disulfide/dithiol center was unaffected by the Lys/ Arg substitution. Considering the structural homologies around the N 5 sites of GR and lipoamide dehydrogenase, these observations clearly reveal the sensitivity of N 5 to its electrostatic environment and the absence of redox communication between N 5 and the disulfide/dithiol center.

Regulation of the Redox Potential of FAD
The functional role of GR is to reduce GSSG from NADPH (Equation 1). Considering that (i) the product of the reaction, GSH, also reduces GR and (ii) the high NADPH/NADP ϩ and GSH/GSSG ratios in the cytoplasm of normal cells, GR is present largely in the EH 2 and MDS states (28).
The present investigation on yeast GR reveals that the structure and environment of the N 10 -C 10a and N 5 ϭC 4a bonds of FAD differ in the EH 2 and MDS species. On one hand, we have observed a steric or electronic influence of the protein on N 10 -C 10a when GR is reduced with NADPH. This effect may be correlated with the slight distortion of the isoalloxazine ring in the NADPH-reduced enzyme. On the other hand, our vibrational data show that the hydrogen bonding state of N 5 is higher for EH 2 generated by GSH reduction than for EH 2 formed by the NADPH action. This different pattern can impose a redox control influencing the catalysis. In particular, the hydrogen bonding state of the N 5 atom influences the redox potential of the flavin, as well as the chemical reactivity of the C 4a site (30,41,42). The modulation of the flavin potentials is due to the fact that N 5 participates with the C 4a , C 10a , and N 1 atoms in the enediamine redox site of the isoalloxazine ring (30). Using a GR mutant lacking the disulfide center, the E m7 value of FAD was estimated to be Ϫ366 mV (6), a value ϳ50 mV more negative than the potential of the NADPH/NADP ϩ couple (Ϫ320 -335 mV) (43)(44)(45). An increased hydrogen bonding interaction at N 5 is expected to stabilize the oxidized state of flavin, thus decreasing the intrinsic redox potential of FAD and making its reduction less favorable (46). In other words, different hydrogen bonding states at N 5 therefore suggest that the redox potential of the flavin ring of GR is indirectly modulated by NADPH and GSH, the intrinsic potential of FAD in the GSH-reduced enzyme being in fact lower than that of the NADPH-reduced enzyme. The high resolution structure of GR indicates that FAD can be equatorially protonated at N 5 (3). A strong hydrogen bond at the N 5 atom of the GSH-reduced enzyme may constitute an energetic barrier that could limit a protonation of N 5 for the formation of reduced FAD.
In conclusion, regulation of the enzyme activity plausibly involves the oxidized FAD of EH 2 and MDS and the reduced forms of the substrates and products of the enzyme, i.e. NADPH and GSH. Our investigation shows that the formation of EH 2 by GSH reinforces the hydrogen bonding state of the N 5 atom. This electrostatic effect likely decreases the intrinsic redox potential of FAD and thus can decrease the efficiency of redox transfer between NADPH and oxidized FAD for the initiation of a new catalytic cycle.