unfolding kinetics of glutathione reductase from cyanobacterium spirulina maxima

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Archives of Biochemistry and BiophysicsVol. 387, No. 2, March 15, pp. 265–272, 2001doi:10.1006/abbi.2000.2230, available online at http://www.idealibrary.com on

Unfolding Kinetics of Glutathione Reductasefrom Cyanobacterium Spirulina maxima

Juan L. Rendon1 and Guillermo Mendoza-HernandezDepartamento de Bioquımica, Facultad de Medicina, Universidad Nacional Autonoma de Mexico,Apartado Postal 70-159 04510 Mexico, D.F.

Received July 25, 2000, and in revised form October 21, 2000; published online February 16, 2001

The kinetics of the irreversible unfolding of gluta-thione reductase (NAD[P]H:GSSG oxidoreductase, EC1.6.4.2.) from cyanobacterium Spirulina maxima wasstudied at pH 7.0 and room temperature. Denaturationwas induced by guanidinium chloride and the changesin enzyme activity, aggregation state, and tertiarystructure were monitored. No full reactivation of en-zyme was obtained, even after very short incubationtimes in the presence of denaturant. Reactivationplots were complex, showing biphasic kinetics. A veryfast early event in the denaturation pathway was thedissociation of tetrameric protein into reactivatablenative-like dimers, followed by its conversion into anonreactivatable intermediary, also dimeric. In thefinal step of the unfolding pathway the latter was dis-sociated into denatured monomers. Fluorescence mea-surements revealed that denaturation of S. maximaglutathione reductase is a slow process. Release of theprostethic group FAD was previous to the unfolding ofthe enzyme. No aggregated species were detected inthe unfolding pathway, dismissing the aggregation ofdenatured polypeptide chains as the origin of irre-versibility. Instead, the transition between the twodimeric intermediates is proposed as the cause of ir-reversibility in the denaturation of S. maxima gluta-hione reductase. A value of 106.6 6 3 kJ mol21 was

obtained for the activation free energy of unfolding inthe absence of denaturant. No evidence for the nativemonomer in the unfolding pathway was obtainedwhich suggests that the dimeric nature of glutathionereductase is essential for the maintenance of the na-tive subunit conformation. © 2001 Academic Press

Key Words: glutathione reductase; denaturation;cyanobacteria; Spirulina.

1
To whom correspondence and reprint requests should be ad-ressed. E-mail: [email protected].
0003-9861/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

The flavoprotein glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) is involved in themaintenance of cellular redox homeostasis (1). Thisenzyme has a wide distribution, both in prokaryoticand eukaryotic organisms (2). Typically, it is a ho-modimeric protein constituted by subunits with an M r

of about 55 kDa. The three-dimensional structure ofglutathione reductase from both Escherichia coli andhuman erythrocyte is well known (3–5). These studieshave revealed that each subunit is arranged into afour-domain tertiary structure (5). The dimeric natureof the enzyme is critical for its function, because bothsubunits contribute with essential residues to the con-stitution of the active site (6). In the particular case ofthe red blood cell enzyme, both subunits are covalentlylinked by a disulfide bond (7).

On the other hand, the quaternary structure of glu-tathione reductase from Spirulina maxima is unusual.In this cyanobacterial species the main form of theenzyme is represented by a tetramer of M r 192 kDa. Atneutral pH and room temperature it coexists in slowequilibrium with a minor dimeric fraction (8). Thethermodynamic aspects of the interconversion, as wellas its dependence on pH, temperature, and phosphateconcentration have been previously analyzed (9–11).Furthermore, the stability of S. maxima glutathionereductase in guanidinium chloride (Gdm-Cl) solutionswas studied (12). Although unfolding of the enzymewas shown to be an irreversible process, the relativelylong incubation times employed in such study must betaken into account. In a similar study, dealing with thedenaturation behavior of human erythrocyte glutathi-one reductase in the presence of Gdm-Cl, a significantdegree of reversibility was found (13). However, shortincubation times (15 min) in Gdm-Cl solutions wereused. It is well known that a long incubation time ofproteins in a denaturing environment can lead to the
formation of conformational isomers characterized by a
265

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266 RENDON AND MENDOZA-HERNANDEZ

wrong configuration of some prolyl peptide bonds (14).For oligomeric proteins, the aggregation of unfoldedpolypeptide chains is a common cause of irreversibility(15).

The lack of information concerning the denaturation/refolding pathway for any glutathione reductase, aswell as the possibility to obtain reversibility after shortincubation times in Gdm-Cl prompted us to performthe present work.

MATERIALS AND METHODS

Materials. All reagents were obtained from Sigma Chemical Co.(St. Louis, MO) and used without further purification. Gdm-Cl (mo-lecular biology grade) was of the highest purity available. Waterpurified by reverse osmosis was used in the preparation of solutions.Unless otherwise noted, all experiments reported in the presentwork were performed in 0.1 M potassium phosphate buffer (pH 7.0)containing 1 mM EDTA (buffer A). In order to avoid pH disturbancesdue to the mixing of Gdm-Cl with the protein, a stock solution of thedenaturant (8 M) was made by dissolving it in the phosphate solu-tion; then, the pH was adjusted to 7.0 by adding KOH from a 10 Nsolution.

Enzyme. Glutathione reductase was purified to homogeneityfrom S. maxima biomass as previously described (8). The purity ofpreparations was routinely assessed by PAGE under denaturingconditions according to Laemmli (16).

Protein determination. The concentration of purified glutathionereductase in the stock solution was determined spectrophotometri-cally at 274 nm (9).

Enzyme assays. Glutathione reductase activity was monitored byfollowing the initial decrease in absorbance at 340 nm as a conse-quence of NADPH oxidation in the presence of GSSG. The reactiva-tion ability of the enzyme as a function of incubation time in thepresence of Gdm-Cl was measured as follows. Denaturation mixtureswere prepared in spectrophotometric cuvettes by mixing S. maximaglutathione reductase (66 ng) with buffer and an adequate volume ofthe Gdm-Cl stock solution in a final volume of 30 ml. At given timentervals, the denaturant was diluted by adding 1 ml of buffer con-aining both GSSG and NADPH, and the reductase activity wasmmediately assayed. Control mixtures were prepared and treateds described but in the absence of Gdm-Cl. For each time point, theelative activity was calculated by taking the corresponding controlctivity as 100%. The maximal residual concentration of the dena-urant in the reactivation experiments was 0.16 M, a condition athich a residual activity greater than 90% of native activity isbtained (12).HPLC gel filtration analysis. Size-exclusion chromatography was

arried out in a SEC 3000 HPLC column (0.75 3 30 cm). All solutionssed for preequilibration and elution were degassed and filteredhrough a Millipore 0.22 mm filter. Prior to injection of the sample,

enzyme was preincubated in buffer A containing the appropriateconcentration of Gdm-Cl. At different time intervals, aliquots (25–30mg protein in 30 ml) were withdrawn and injected onto the chroma-tography column. The elution profiles were monitored by recordingthe absorbance at 280 nm. Chromatograms were collected in a com-puter. For each incubation time, the area under the correspondingcurve was obtained by curve fitting of the chromatogram with theprogram Peak Fit (Jandel Scientific). For Stokes radius (RS) estima-tion, the column was calibrated according to Laurent and Killander(17), using well characterized protein standards.

Crosslinking experiments. A combination of covalent crosslinkingand electrophoresis under denaturing conditions was used to analyze
the oligomeric nature of the intermediates in the unfolding pathway.Enzyme aliquots (80 mg protein) were mixed with an adequate vol-
me of the Gdm-Cl stock solution to give the desired concentration infinal volume of 250 ml. After a 14-h incubation, a 20-ml aliquot of

5% glutardialdehyde was added and the crosslinking reaction wasllowed to continue for 1 min. Then a small volume (30 ml) of 2 M

NaBH4 was added to inactivate the excess of glutardialdehyde. Thecrosslinking time was optimized in the course of our calorimetricalstudies (11). After 20 min, denaturant was removed by passing theincubation mixtures through a Sephadex G-25 column (15 3 1.6 cm);then the eluted solutions were concentrated to the original volumesand the protein was recovered by adding 20 ml of 10% sodium

eoxycholate followed by 30 ml of 60% trichloroacetic acid. Remotionof the denaturant was a necessary additional step due to the incom-patibility between Gdm-Cl and deoxycholate.

Fluorescence measurements. Gross conformational changes con-comitant with unfolding were monitored by following the changes inintensity of the intrinsic fluorescence of the protein in an AmincoSPF-500 spectrofluorometer. Samples containing a fixed amount ofglutathione reductase (20 mg) were added under continuous stirringto the fluorometric cell containing the corresponding Gdm-Cl concen-tration in a final volume of 1 ml. Unfolding of the enzyme wasmonitored by following the increase in fluorescence intensity at 354nm. The selected wavelength represents the maximal difference inthe fluorescence emission between the native and the denaturedstates of S. maxima glutathione reductase (12), thus allowing tofollow the kinetics of the transition with the maximal sensitivity.

RESULTS

Reactivation kinetics. Figure 1a shows the loss ofthe reactivation ability of S. maxima glutathione re-ductase as a function of incubation time at 4, 4.5, 5, and5.5 M Gdm-Cl. The choice of these Gdm-Cl concentra-tions was based on a previous work (12); they representthe conditions where a full irreversibility of enzymedenaturation was achieved. It is evident that even atshort incubation times, the recovery of the enzymaticactivity was only partial, irrespective of the Gdm-Clconcentration. The possibility that the reductase activ-ity could be fully recovered by prolonged incubationafter diluting out the denaturant, was analyzed asfollows. Denaturing mixtures at 5 M Gdm-Cl wereprepared and treated as in Fig. 1a. After dilution atselected times, samples were incubated for a 24 h pe-riod before the enzymatic activity was assayed. Theresultant reactivation percentage was essentially iden-tical to that shown in Fig. 1a at the same Gdn-Clconcentration (data not shown). The same experiment,but in which the dilution was performed in the pres-ence of additional FAD, resulted similarly in no gain inreactivation. It should be noted that an analysis ofresidual activity in the presence of the same concen-trations of Gdm-Cl used in the experiments of Fig. 1could not be performed, because even at very shortincubation times, no enzymatic activity remains. Thus,the activity obtained in the reactivation experimentsmust be assigned to a species, which is able to bereactivated. On the other hand, the profile rate atwhich glutathione reductase is irreversibly inactivatedis not simple. When the data were plotted in a semi-logarithmic fashion, the existence of two kinetic phases
under all conditions tested was revealed (Fig. 1b). In

267DENATURATION KINETICS OF GLUTATHIONE REDUCTASE

order to estimate the magnitude of the specific rateconstants, data of Fig. 1a were fitted into a doubleexponential decay function:

A (t) 5 A1e (2k1t) 1 A2e (2k2t) [1]

where A (t) represents the activity at time t; A 1 and A 2

are the amplitudes of the change for the fast and slowphases, respectively, and k 1 and k 2 represent the cor-responding first-order rate constant. In Table I the

FIG. 1. Reactivation kinetics of glutathione reductase as a functionof incubation time in Gdm-Cl solutions. (A) Time courses of therelative activity observed immediately after dilution of denaturant.Each point represents the average value of seven independent ex-periments. The corresponding concentrations of Gdm are: (E) 4 M;(h) 4.5 M; (F) 5 M; (‚) 5.5 M. (B) Semilogarithmic plot of the dataobtained at 5 M Gdn-Cl showing the biphasic nature of the irrevers-ible inactivation. (F) Experimental data; (E) secondary plot showingthe fast phase, obtained by substracting the extrapolated values(dashed line) of the linear portion from the experimental values.

resultant values of the fit are shown.

Hydrodynamic analysis. The changes in the aggre-gation state of the enzyme, concomitant with its irre-versible inactivation, were analyzed at 4 and 5 MGdm-Cl by HPLC as described under Materials andMethods. Figure 2 shows the elution patterns as afunction of incubation time in the presence of 4 MGdm-Cl. After a 7-min contact with the denaturant, amajor species (peak II), centered at about 8.3 min, wasobtained (due to the time between the injection of thesample and its appearance in the eluted volume, theshortest time to analyze was 7 min). From the calibra-tion curve, a RS of 3.8 6 0.4 nm was calculated for peakII. Two minor peaks, with retention times of 7.6 and10.95 min, were also detected. The elution volume cor-responding to the latter is almost coincident with thetotal volume of the column, which suggested a lowmolecular weight component, probably the prostheticgroup FAD. With regard to the 7.6 min species (peak I),it was characterized by a RS of 5.4 6 0.4 nm.

TABLE I

Parameters Obtained from Adjustment of ReactivationData to Equation 1

[Gdm-Cl] A 1 (%) k 1 (s21) A 2 (%) k 2 (s21)

4 21.89 8.33 3 1023 77.1 1.15 3 1024

4.5 49.4 1.83 3 1023 47.6 1.92 3 1024

5 39.4 9.17 3 1023 61.2 1.25 3 1023

5.5 68.1 15 3 1023 28.2 5.8 3 1023

FIG. 2. Chromatographic profiles of glutathione reductase as afunction of incubation time at 4 M Gdm-Cl. Size exclusion chroma-tography was performed as described under Materials and Methods.The labeled peaks refer to: (T) native tetramer; (D) native dimer; (I)unfolded monomer; and (II) dimer-like intermediates. The profilemarked “C” represents the chromatographic elution pattern in the
absence of Gdm-Cl. For clarity, the peak corresponding to free FADwas omitted.

1


Longer incubation times in 4 M Gdm-Cl resulted insuch changes in the relative proportions of both proteinforms, that peak I increased in relative abundanceconcomitant with a decrease of peak II. Figure 3 showsthese changes as a function of incubation time in 4 MGdm-Cl. Data were fitted to a single exponential equa-tion and the corresponding rate constant was esti-mated. Interestingly, no change in the retention timeof both peaks with incubation time was observed. At 5M Gdm-Cl, a similar pattern of changes was observed,but at a higher rate. However, it is interesting to notean increase in the RS, from 5.4 to 6.1 nm, of peak I. Thisobservation suggests a less compact state for this spe-cies.

In order to get insight into the oligomeric nature ofthe chromatographic species, enzyme samples werepreincubated with different concentrations of Gdm-Cland crosslinked with glutardialdehyde as describedpreviously. Figure 4 shows the resulting electro-phoretic patterns along with the elution pattern of theenzyme after 14 h incubation at 4 or 5 M Gdm-Cl. Froma comparison of the relative intensities of both theprotein bands and the chromatographic zones, it be-comes clear that peak I and peak II correspond tomonomeric and dimeric forms, respectively, of the en-zyme. Their conformational status will be discussedbelow.

Incubation times longer than 3.5 h at 4 M Gdm-Clresulted in the appearance of a minor species whoseretention time was lower than that of the 7.6 min

FIG. 3. Time course of the relative abundance of the protein peaksand free FAD at 4 M Gdm-Cl. For each time, the correspondingchromatographic profile was analyzed as described under Materialsand Methods. Solid line represents the best fit into a first orderprocess. The corresponding first-order rate constants were: 3.3 31025 s21 for monomer (F); 2.1 3 1025 s21 for dimer (E), and 11.5 3025 s21 for free FAD (Œ).

species (Fig. 4B); however, due to its late appearance,

no further attempt to characterize this species wasmade.

Release of FAD during unfolding. As mentionedabove, the appearance and increase of a chromato-graphic peak whose retention time suggested a lowmolecular weight species was observed in all gel filtra-tion experiments, both at 4 and 5 M Gdm-Cl. Themolecular nature of this species was confirmed throughspectroscopic analysis. The resultant visible spectrumwas coincident with that of oxidized flavine, stronglysuggesting the presence of the prosthetic group FAD.Like the changes observed in the relative abundance ofthe protein species, the increase in concentration offree FAD followed simple exponential kinetics (Fig. 3).Although at 5 M Gdm-Cl the specific rate constant forthe appearance of free FAD (1.28 3 1023 s21) wasalmost coincident with that corresponding to the in-crease of monomer abundance (1.2 3 1023 s21), at 4 Mof the denaturant the release of FAD was a faster

FIG. 4. (A) Electrophoretic pattern of crosslinked samples obtainedafter long-term incubation in Gdm-Cl solutions. Enzyme aliquotswere incubated for 14 h in the presence of the corresponding Gdm-Clconcentration. Then glutardialdehyde was added and the sampleswere processed as described under Materials and Methods. (A) 4 MGdm-Cl; (B) 4.5 M Gdm-Cl; (C) 5 M Gdm-Cl; (D) noncrosslinkedenzyme; (E) molecular weight markers. (B) Chromatographic profiles
of enzyme after 14 h incubation in the presence of either 4 M Gdm-Clor 5 M Gdm-Cl.

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process as compared with the monomer appearance(Fig. 3).

Conformational changes. Changes in the tertiarystructure of S. maxima glutathione reductase withtime were recorded by following the increase in inten-sity of the fluorescence emission at 354 nm (Fig. 5). Inspite of the complex oligomeric nature of the enzyme, asingle and slow transition was observed at all theGdm-Cl concentrations tested. Attempts to detect afast conformational change by stopped-flow fluores-cence measurements were unsuccessful under all con-ditions. Hence, fluorescence data were fitted to themonophasic equation:

Ft 5 FO 1 ~F` 2 FO!~1 2 e 2kt!, [2]

where F o and F` represent the initial and the maximalvalues in fluorescence intensity, and F t is the fluores-ence value at time t. From the slope of the logarithmicorm of the equation, the value of the correspondingnfolding rate constant ku was obtained.Activation energy analysis. Due to the irreversible

nature of the conformational transition of S. maximaglutathione reductase, the specific rate constants cal-culated from fluorescence measurements representtrue unfolding rate constants. The Gdm-Cl dependenceof the latter was fitted to Eq. [3], thus enabling thedetermination of the unfolding rate constant in theabsence of denaturant:

FIG. 5. Progress curves of unfolding for glutathione reductase de-ected by fluorescence measurements. The unfolding reaction wastarted by a jump from 0 to the corresponding concentration ofdm-Cl and the increase in the fluorescence emission intensity at54 nm was recorded. The continuous lines represent the best fittingf data to Eq. [2] of the text. Symbols are: (h) 4 M Gdm-Cl; (%) 4.5 M

Gdm-Cl; (‚) 5 M Gdm-Cl; (F) 5.5 M Gdm-Cl.

log ku 5 log ku~H2O! 1 mu@Gdm-Cl#, [3]

where ku is the apparent first-order rate constant forunfolding at different concentrations of denaturant, ku

(H2O) is the apparent unfolding rate constant in theabsence of denaturant, and mu, the slope of the graph,is interpreted as the change in solvent accessibility ofthe unfolded state (18). Figure 6 shows the resultantplot for S. maxima glutathione reductase. By extrapo-ating to zero concentration of Gdm-Cl, a value of 6 3027 s21 was obtained for ku (H2O). From this, the free

energy of activation for unfolding (DG‡) was calculatedfrom the Eyring equation:

ku 5 kB

Th expS2

DG ‡

RT D , [4]

where kB and h are the Boltzmann and Planck con-stants, respectively; T is the absolute temperature; andR is the universal gas constant (19). The correspondingvalue of DG ‡ was 106.6 6 3 kJ mol21.

DISCUSSION

Studies dealing with the stability of oligomeric pro-teins have led to information about its intrinsic stabil-ity (20), and the role that the subunit interaction playsin the maintenance of a native conformation of sub-units (21). For glutathione reductase, systematic stud-ies dealing with stability are scarce (12, 13), in spite ofthe great detail at which its tertiary structure is known(5, 6). In denaturation–renaturation experiments per-formed under equilibrium conditions, a partial reacti-vation under optimal conditions (;65%) was found forhe enzyme from human red blood cells (13). By con-rast, no reversibility was obtained for glutathione re-uctase from the cyanobacterium S. maxima (12).

FIG. 6. Gdm-Cl dependence of the logarithm of the unfolding rateconstants for glutathione reductase. Each point was obtained fromthe fluorescence data of Fig. 5 and represents the average of three
independent experiments. The solid line was obtained by fittingexperimental data into Eq. [3] (r 5 0.97).

mc

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However, two factors that potentially could contributeto explain such different behavior must be taken intoaccount. First, the different times of incubation in thepresence of the denaturant; while for the cyanobacte-rial enzyme the measurements of physicochemical pa-rameters were performed after 24 h incubation in theGdm-Cl solutions (12), for human glutathione reduc-tase a very short time (15 min) was used (13). Second,the oligomeric nature of both proteins; as mentionedpreviously, glutathione reductase of S. maxima is

ainly a tetrameric enzyme (8, 9), while its humanounterpart exists as a dimer (4).

In the present work we characterized the time coursef the major structural changes that are associatedith the irreversible unfolding of glutathione reduc-

ase from the cyanobacterium S. maxima in Gdm-Clolutions. As shown in Fig. 1, no full reversibility wasbserved when enzymatic activity was measured im-ediately after dilution of Gdm-Cl, even at very short

ncubation times under denaturing conditions. For var-ous oligomeric proteins, the rate-limiting step of fold-ng is determined by subunit association, a processequiring minutes to hours (15). However, in the casef S. maxima glutathione reductase, this possibilityas excluded because no increase in reactivation wasbserved after 24 h of dilution of the denaturant. Fur-hermore, the total inhibition of the enzyme in theresence of Gdm-Cl, and therefore the absolute lack ofesidual activity, strongly suggest the absence of anyorm of native enzyme. Although the absence of resid-al activity could simply reflect the inability of theubstrates to form a productive complex, such inabilityust be caused by a Gdm-Cl induced conformational

hange, particularly when the high concentrations ofdm-Cl used in the present study are taken into ac-

ount. It is a well-known fact that those regions of thenzymes involved in the binding and catalysis of sub-trates (e.g., the active site) are characterized by areat conformational flexibility (22, 23), showing aigher susceptibility to denaturants. Hence, the rela-ive activities shown in Fig. 1 were due neither toesidual native enzyme nor to the refolding of dena-ured monomers and its association but to a speciesble to be reactivated. Moreover, this species is in theimeric aggregation state. This conclusion was basedn a comparison of the reactivation data with the gelltration experiments, which show the total absence ofetrameric glutathione reductase and the presence of aimer-like conformer, in coexistence with a monomeropulation. The relative abundance of both species isependent on incubation time in the presence of Gdm-l. However, it is evident that the monomer detectednder all conditions do not correspond to the nativeonformation of subunit. This conclusion was based onhe following facts: (a) its R (5.4 nm) is far above the
S
expected RS for a compact monomer of 48 kDa (;3.1 w

m), and (b) the rate at which the fluorescence emis-ion intensity increase suggest gross conformationalhanges are ocurring concomitantly with monomer ap-earance.On the other hand, there is an inverse relationship

etween the abundance of the monomeric fraction andhe ability of enzyme for full reactivation, although it isorth noting that either at 4 or 5 M Gdm-Cl, the

rreversible inactivation was always higher than theredicted value from the amount of denatured mono-er. In Fig. 7 the relative changes in the irreversible

nactivation at 4 M Gdm-Cl are compared with theorresponding increase in abundance for both unfoldedonomer and free FAD in the time interval at which

eactivation measurements were carried out; it is evi-ent a close correlation between the irreversible loss inctivity and the appearance of free FAD, while thencrease in the denatured monomer occurs at a lowerate. Even at 5 M Gdm-Cl, the percentage of irrevers-ble inhibition was higher as compared with the gain ofenatured monomer (data not shown). Thus, a signifi-ative fraction of the irreversible inactivation must bettributable to intermediates other than the denaturedonomer, leading to the conclusion that the dimeric

raction observed in the chromatographic experimentss composed of two distinct populations: a fully reacti-atable conformer, which is irreversibly converted intonon-reactivatable intermediate. The latter is charac-

erized by the loss of the prosthetic group. Such irre-ersible transition between these two conformationalsomers could explain the fast phase of the reactivationlots shown in Fig. 1.Although the increase in free FAD is concomitant

FIG. 7. Relative changes in time of the irreversible inactivation,free FAD and unfolded monomer of glutathione reductase at 4 MGdm-Cl. Irreversible inactivation (F) was calculated by substractingthe corresponding reactivation data of Fig. 1 from control activity.Data for both free FAD (‚) and unfolded monomer (E) were obtainedfrom its elution profiles of the chromatographic experiments.

ith the irreversible inactivation, such parallelism is

bta


the result of the inability of the nonreactivatable inter-mediate to recover the prosthetic group due to an irre-versible conformational change. On the other hand, theabsence of any tetrameric fraction in the chromato-graphic experiments suggests that its dissociation intothe reactivatable dimer-like intermediate is a veryearly process in the unfolding pathway.

The transition from the nonreactivatable dimericspecies into the denatured monomer involves simulta-neous changes in the tertiary and quaternary struc-tures. The slowness of these processes allowed a de-tailed characterization. Interestingly, neither a nativenor a structured monomer was detected in the unfold-ing pathway, indicating an intrinsic unstability for thisspecies and suggesting that the dimeric nature of glu-tathione reductase is essential for the maintenance ofthe native conformation of the subunit.

It is worth noting that, in spite of the complex do-main structure of glutathione reductase (4), the kinetictransition between the nonreactivatable dimeric inter-mediate and the denatured state is a simple one, thusrevealing a high degree of cooperativity in the unfold-ing of domains. This feature is a common trait for bothhuman (13) and S. maxima glutathione reductase, al-beit the cyanobacterial enzyme is denatured fifteentimes slower (ku 5 2.6 3 1023 s21) than its humancounterpart (ku 5 4 3 1022 s21) at 5 M Gdm-Cl. Ingeneral, the rate of unfolding of a protein is inverselyproportional to the stability of its native state (14).Therefore, it can be concluded that glutathione reduc-tase from S. maxima shows a higher stability as con-trasted with the human enzyme.

A comparison of our results with other proteins forwhich denaturation kinetic data are available (24–26),reveals that the native 3 unfolded transition of S.maxima glutathione reductase is a slow process, evenin the presence of relatively high concentrations ofGdm-Cl. The estimated value for the activation freeenergy of unfolding under native conditions (DG7 5107 6 3 kJ mol21) is consistent with the observedslowness of the unfolding process.

Based on the experimental results, we propose thefollowing kinetic model in order to explain the irrevers-ibility of S. maxima glutathione reductase denatur-ation:

M4 ^ 2M*2 ¡

k f

2M**2 ¡

ks

4D,

where M 4 represents the native tetramer. Although inthe absence of any perturbing reagent such speciescoexists in a slow equilibrium with a dimeric fraction(8, 9), the addition of Gdm-Cl at the concentrations
used in the present study results in a full and fast
displacement of equilibrium toward a dimeric interme-diate which is able to be reactivated (M*2), followed bythe irreversible transition into a nonreactivatable spe-cies, also dimeric (M**2). The existence of M*2 in theunfolding pathway of S. maxima glutathione reductaseis proposed on the basis of the reactivation experi-ments shown in Fig. 1, as discussed above. After thedilution of Gdm-Cl, the remaining dimeric intermedi-ate M*2 is reverted into active species, being responsi-le for the observed reactivation; therefore, for anyime the relative abundance of the dimeric intermedi-ry M*2 is given by the percentage of reactivation. On

the other hand, the M**2 species is characterized by theloss of the prosthetic group FAD due to an irreversibleconformational change, rendering it fully inactive.Both M*2 and M**2 are conformationally distinct, buthydrodynamically indistinguishable from the nativedimer. The proposed dimeric nature for both interme-diates was confirmed by electrophoretic experimentson cross-linked samples (Fig. 4). We propose that theconversion of M*2 into M**2 represents the step involvedin the observed irreversibility, due to an early confor-mational change. The fast phase observed in the reac-tivation experiments is related to such irreversibletransition. In the final step of the unfolding pathway,the kinetic intermediary M**2 dissociates and unfoldsinto the denatured monomer (D), an irreversible andslow process. The activation free energy of unfolding,calculated from the fluorescence emission measure-ments, is attributable to this last step. From the inac-tivation and chromatographic data summarized in Fig.7, it becomes clear that the irreversible inactivation isgiven by the relative abundance of both M**2 and thedenatured monomer.

The lack of information regarding the structural sta-bility of other disulfide oxidoreductases, prevent usfrom comparing the denaturation behavior of S. max-ima glutathione reductase with that of homologousproteins.

ACKNOWLEDGMENT

This work was supported by Research Grant IN 202397 fromDireccion General de Asuntos del Personal Academico (DGAPA),UNAM.

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