Proc. Nati Acad. Sci. USA 79 (1982)

Correction. In the article "Possible occurrence and role of anessential histidyl residue in succinate dehydrogenase" by Ste-ven B. Vik and Youssef Hatefi, which appeared in the Novem-ber 1981 issue of Proc. Nati Acad. Sci. USA (78, 6749-6753),two undefined acronyms appear on pages 6750 and 6751. Oneis DCIP and should be C12IP; the other is DEPC and shouldbe Et2PC.

Correction. In the article "Intracellular hormone receptors:Evidence for insulin and lactogen receptors in a unique vesiclesedimenting in lysosome fractions of rat liver" by Masood N.Khan, Barry I. Posner, Anil K. Verma, Rahat J. Khan, and JohnJ. M. Bergeron, which appeared in the August 1981 issue ofProc. Nati Acad. Sci. USA (78, 4980-4984), two panels of Fig.4 were described incorrectly in the legend. The correct descrip-tion is that, in Fig. 4, B represents somatotropin binding sitesand C represents insulin binding sites.

Correction. In the article "Isolation and amino acid sequenceof a morphogenetic peptide from hydra" by H. Chica Schallerand Heinz Bodenmuller, which appeared in the November1981 issue of Proc. Natl. Acad. Sci. USA (78, 7000-7004), anerror by the printer resulted in Fig. 6 being printed upsidedown and reversed from side to side. The correct picture isshown below.

Correction. In the article "Multiple differences between thenucleic acid sequences of the IgG2aa and IgG2ab alleles of themouse" by Peter H. Schreier, Alfred L. M. Bothwell, BennoMueller-Hill, and David Baltimore, which appeared in the July1981 issue of Proc. NatL Acad. Sci. USA (78, 4495-4499), theauthors request that the following correction be noted. The lastline of the Abstract should read "from the IgG2aa allele by theIgG2ba allele."

Correction. In the article "Molecular basis for familial isolatedgrowth hormone deficiency" by John A. Phillips III, Brian L.Hjelle, Peter H. Seeburg, and Milo Zachmann, which appearedin the October 1981 issue of Proc. NatL Acad. Sci. USA (78,6372-6375), an undetected printer's error occurred on p. 6372.Fig. 1 was printed upside down. It and its legend are repro-duced correctly here.

I .

FIG. 1. Pedigrees of families I-rn, each of which has one or moreindividuals affected with IGHD type A (a, i).

1336 Corrections

Proc. Natl Acad. Sci. USAVol. 78, No. 11, pp. 6749-6753, November 1981Biochemistry

Possible occurrence and role of an essential histidyl residue insuccinate dehydrogenase

(active site/mechanisms of succinate oxidation and fumarate reduction)

STEVEN B. VIK AND YOUSSEF HATEFI*Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

Communicated by David E. Green, July 27, 1981

ABSTRACT Diethylpyrocarbonate (E4PC) inhibits the suc-cinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC1.3.99.1] activity of submitochondrial particles, Complex II (suc-cinate:ubiquinone oxidoreductase), and the soluble, pure succi-nate dehydrogenase. The reaction order with respect to Et2PCconcentration is close to unity, suggesting modification of one es-sential residue per active unit of the enzyme. The pH profile ofEt2PC inhibition, the partial reversal of inhibition by hydroxyl-amine, and the spectral change of the E4PC-treated enzyme inthe UV region suggest modification ofa histidyl residue. Succinatedehydrogenase activity can be protected against Et2PC inhibitionby succinate, fumarate, malonate, or oxaloacetate (also by acti-vating anions such as ClOi and Br-), suggesting that the Et2PC-modified essential residue might be at the active site. In both sub-mitochondrial particles and the purified enzyme, succinate de-hydrogenase activity is highest and relatively constant at pH > 7.0and diminishes precipitously at pH < 7.0. By contrast, fumaratereductase activity is highest at pH c 7.0 and diminishes at pH> 7.0. These results are consistent with the possible participationofthe unprotonated and protonated forms ofthe imidazole moietyofthe putative histidyl residue, respectively, in succinate oxidationand fumarate reduction.

Bovine heart succinate dehydrogenase [succinate:(acceptor) ox-idoreductase, EC 1.3.99.1], purified in 1971, is an iron-sulfurflavoprotein composed of two unequal subunits (1-3). Thelarger subunit has a molecular weight of70,000 and contains permol 1 mol of covalently bound FAD, 4 mol of Fe, and 4 mol oflabile sulfide. The smaller subunit has a molecular weight of27,000 and contains per mol 3-4 mol ofFe and 3-4 mol oflabilesulfide (1, 3). EPR studies have suggested the presence of twobinuclear and one tetranuclear iron-sulfur centers in succinatedehydrogenase (4). Succinate dehydrogenase contains an es-sential sulfhydryl group, apparently located in the large subunitat the active site (5, 6). The mechanism of succinate oxidationand the role of the essential thiol in enzyme activity are notknown. It has been suggested (5-7) and disputed (8) that theessential thiol is involved in the tight binding of the inhibitor,oxaloacetate. Dehydrogenation of succinate by the enzyme istrans, and the two pairs of trans hydrogens are equivalent inthis regard (9, 10). Monohalogen-substituted succinate analogsare also oxidized by succinate dehydrogenase, but only in theL-configuration (11).The studies reported below have indicated the possible pres-

ence of an essential histidyl residue at the enzyme active site.pH studies of the modification of this residue by diethylpyro-carbonate (Et2PC) and of enzyme activity during succinate ox-idation and fumarate reduction have suggested that the imida-zole moiety of the putative essential histidyl residue might

participate as a base in succinate oxidation and as a conjugateacid in fumarate reduction.

METHODS AND MATERIALSSubmitochondrial particles (SMP) (12), Complex II (suc-cinate:ubiquinone oxidoreductase; ref. 13), and dehydrogenase(1) were prepared and assayed for succinate dehydrogenase (1,13), succinate:ubiquinone oxidoreductase (13), and succinateoxidase (2) activities as before. SMP were suspended at 8 mg/ml in 0.25 M sucrose/100 mM potassium phosphate at pH 6.0or at pH values otherwise indicated. The particles were acti-vated by a 10-min incubation at 38°C and cooled on ice beforeuse. Stock Et2PC was freshly diluted to a concentration of 0.55M in absolute ethanol, an aliquot to give the indicated concen-trations was added to the SMP suspension, and the mixture wasincubated at 0°C and sampled for activity assay. Where indi-cated, the protecting agents (i.e., succinate, fumarate, malo-nate, NaClO4, NaBr) were added at the stated concentrationsto the SMP suspension prior to the addition of Et2PC. Proteinwas determined by the biuret method (14) for SMP in the pres-ence of 1 mg ofdeoxycholate per ml and by the method ofLowryet al. (15) for Complex II and succinate dehydrogenase.

Protection by oxaloacetate against inhibition of SMP byEt2PC was carried out as follows. The SMP suspension (8 mg/ml) in sucrose/phosphate buffer was divided into two parts.One part was treated with 2.7 mM Et2PC for 20 min at 0°C andthen centrifuged for 15 min at 80,000 x g. The pellet was sus-pended in the sucrose/phosphate buffer supplemented with 20mM succinate, incubated for activation for 30 min at 38°C, andassayed for succinate:phenazine methosulfate (PMS)/2,6-di-chloroindophenol (Cl2IP) oxidoreductase activity. The secondaliquot of SMP was treated with 2.5 mM oxaloacetate, allowedto stand several minutes at 0°C, centrifuged, resuspended inthe sucrose/phosphate buffer at 8 mg/ml, and divided into twoparts. One part was activated in the presence of 20 mM suc-cinate for 30 min at 38°C and assayed. The second part wastreated with 2.7 mM Et2PC for 20 min at 0°C, centrifuged, re-suspended in the sucrose/phosphate buffer, activated in thepresence of 20 mM succinate, and assayed for activity.

Reactivation of Et2PC-treated SMP by hydroxylamine wascarried out as follows. The SMP suspension (8 mg/ml) in 0.25M sucrose/100 mM potassium phosphate, pH 6.0, was incu-bated for 10 min at 38°C, cooled on ice, and assayed for suc-cinate:PMS/Cl2IP oxidoreductase activity. The suspension wasdivided into two 12-ml parts, and one part was treated at 0°Cwith 5 1,u of6.9 M Et2PC (final Et2PC concentration, 2.87 mM).Both fractions were periodically assayed for activity until the

Abbreviations: SMP, submitochondrial particles; Et2PC, diethyl py-rocarbonate; PMS, phenazine methosulfate; Cl2IP, 2,6-dichlo-roindophenol.* To whom reprint requests should be addressed.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

6749

6750 Biochemistry: Vik and Hatefi

Et2PC-treated fraction was inhibited by 2 50%. One ml of0.59M histidine was added to each fraction to stop further Et2PCinhibition, and the suspensions were centrifuged for 15 min at80,000 X g to remove excess Et2PC. The pellets were sus-

pended in sucrose/phosphate buffer, pH 7.0, and assayed foractivity. The Et2PC-treated fraction was divided into four parts,and each part was treated with a different volume of 3.0 M hy-droxylamine to give the concentrations indicated in Table 2. Thecontrol SMP fraction was similarly treated. After 30 or 60 minofincubation at room temperature, all samples were centrifugedas above to remove excess hydroxylamine. They were then sus-

pended in sucrose/phosphate buffer, pH 7.0, and assayed forprotein and succinate:PMS/DCIP oxidoreductase activity.After hydroxylamine was added to the Et2PC-treated ComplexII of Fig. 3 and the spectral change was recorded, it was thenremoved by the centrifuge column method of Penefsky (16)before assay of Complex II for activity. Removal of hydroxyl-amine was necessary because it interfered with activity assays

by reducing Cl2IP.Assays for fumarate reduction were conducted as follows.

When the enzyme was succinate dehydrogenase, the reactionmixture (final vol, 3.0 ml) contained 50 mM potassium phos-phate at the pH values indicated, 10mM EDTA, 60 uM pheno-safranine, and 10 mM fumarate. When the enzyme source wasSMP, the reaction mixture contained in addition 0.25 M sucrose

and 1.7 mM NaN3. The mixture was placed in an anaerobiccuvette fitted with a rubber stopper and deaerated by aspiration.Then 0.5 M sodium dithionite in 1 M Tris HCl (pH 7.4) was

added through a microsyringe until the purple color of the dyefaded. Normally, about 300,uM dithionite was required, andthe Tris added together with dithionite did not alter the pH ofthe reaction mixture. After complete reduction of phenosafra-nine, some air was admitted to the cuvette and the mixture was

shaken until the dye began to become reoxidized and the so-

lution became lightly pink. This could be done easily, and thereoxidation of the dye as determined spectrophotometricallycould be controlled to <10 %. The cuvette was then evacuatedand placed in the spectrophotometer for temperature equili-bration (38°C); the absorbance was recorded at 520 nm. Afterseveral minutes, during which the reduction state of the dyewas ascertained to be constant, the enzyme was added througha microsyringe in a small volume, and phenosafranine oxidationwas recorded as a function of time at 520 nm. In the absenceof fumarate in the reaction mixture, enzyme addition did notresult in a significant change in absorbance. The A520 of phen-osafranine (oxidized minus reduced) used for calculation of ac-

tivities was 40 X 103 MW1 cm-'. All specific activities are ex-

pressed as,umol of succinate oxidized or fumarate reduced per

min per mg of protein.Et2PC, hydroxylamine, PMS, p-chloromercuriphenyl sul-

fonate, egg lysolecithin, sodium succinate, and sodium oxaloac-etate were obtained from Sigma, C12IP was from Eastman, andsodium fumarate was from Aldrich. Ubiquinone 10 (coenzymeQ2) was a gift from S. Osono (Eisai Co., Ltd., Tokyo).

RESULTSFig. 1 shows semiogarithmic plots of succinate:PMS/Cl2IPox-idoreductase activity of SMP as a function of the duration of in-cubation in the presence of 1.4, 2.7 and 5.5 mM Et2PC; theInset shows the second-order inhibition rate constant (12M-min-1). Similar results were obtained for the effect ofEt2PCon the succinate:ubiquinone oxidoreductase activity of SMP.Et2PC also inhibited the succinate:PMS/DCIP andsuccinate:ubiquinone oxidoreductase activities of Complex IIand the succinate:PMS/DCIP oxidoreductase activity of puri-fied succinate dehydrogenase. Plots of the logarithms of inhi-

-2.0

10 20 30 40 50Minutes

FIG. 1. Semilogarithmic plots of the inhibition time course of suc-cinate:PMS/Cl2IP oxidoreductase activity of SMP in the presence of1.4 (A), 2.7 (o), and 5.5 (v) mM Et2PC. Control rates in the absence ofEt2PC remained unchanged. VO, rate at zero time in the absence ofEt2PC; V, rates at the times indicated in the presence of Et2PC. (Inset)Plot of pseudo-first-order inhibition rate constant k vs. Et2PC concen-tration gives a second-order rate constant of 12 M-1 min-.

bition rate constants of succinate:PMS/DCIP andsuccinate:ubiquinone oxidoreductase activities against the log-arithms of the corresponding Et2PC concentations (Fig. 2)showed slopes near unity, thus indicating that activity inhibitionby Et2PC involved modification of one essential residue onevery active unit of the enzyme. Succinate, fumarate, and theinhibitors malonate and oxaloacetate protected the enzymeagainst inhibition by Et2PC (Table 1). Furthermore, ions suchas ClOO1 and Br-, which are known to activate succinate de-hydrogenase, offered partial protection (Table 1). In these stud-ies, incubation of the enzyme with Et2PC was carried out at pH6.0, a condition that favors Et2PC modification of histidyl res-idues as compared to other residues (e.g., lysyl, tyrosyl, cys-teinyl, and arginyl) modifiable by this reagent. However, theproduct of Et2PC interaction with the imidazole groups (i.e.,N-ethoxyformylimidazole) is cleavable by hydroxylanine (17,18). This reversal is considered to be rather specific for imida-zole groups (19). Table 2 shows that the Et2PC inhibition ofsuccinate dehydrogenase activity ofSMP was partially reversed

-1.2

-1.4

_u

-1.61

-1.81

0.2 0.4 0.6log (mM DEPC)

0.8

FIG. 2. Plots of log k versus log Et2PC concentration from the dataof Fig. 1 (o) and a similar experiment for inhibition of succi-nate:ubiquinone oxidoreductase activity of SMP (0). The slopes of thelines show the reaction orders with respect to Et2PC concentration:o--o, 1.17;*-*, 0.91.

./!01-1.

I~~~~-1.8- I

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. NatL Acad. Sci. USA 78 (1981) 6751

Table 1. Protection of succinate dehydrogenase activity bysuccinate, fumarate, malonate, oxaloacetate and activating anionsagainst Et2PC inhibition*

Exp. Protecting agents k, min-1 None 0.117

Succinate 0.019Fumarate 0.019Malonate 0.014

2 None 0.141NaBr 0.064NaClO4 0.045

Conditions Specific Activity3 SMP 1.1

SMP + Et2PC 0.11OA-SMP 0.63OA-SMP + Et2PC 0.43

In experiments 1 and 2, the concentrations of protective agents were:succinate, 5 mM; fumarate, 5 mM, malonate, 5mM; NaBr, 200mM; andNaCl04, 50 mM. The Et2PC concentration was 5.5 mM. In experiment2, 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0, was used asbuffer instead of 100mM potassium phosphate. OA-SMP, SMPtreatedwith oxaloacetate; k, pseudofirst order inhibition rate constant; spe-cific activity, ,umol of succinate oxidized by PMS/C12IP per min permg of SMP protein.

upon subsequent incubation of the particles with hydroxyl-amine. Partial reversal by hydroxylamine is not uncommon be-cause irreversible disubstituted histidyl derivatives can formeven at moderate Et2PC concentrations (17). A second char-acteristic ofEt2PC modified histidine is an absorption band witha maximum at 234-236 nm, which changes, however, by severalnanometers in various proteins (17, 20). The difference spec-trum of Complex II treated with Et2PC showed an absorptionband with a maximum at 236 nm (Fig. 3, trace 2). At this stage,succinate dehydrogenase activity ofComplex II was completelyinhibited. When the enzyme was further treated with hydrox-ylamine, the absorption intensity decreased and the maximumshifted somewhat toward higher wavelengths (Fig. 3, trace 3).These latter spectral changes were accompanied by partial re-covery of succinate dehydrogenase activity. The negative ab-sorbance change in trace 2, which was also reversed by hy-droxylamine, suggests that in addition to a histidyl residue, atyrosyl residue also might have been modified (17). Finally, theeffect ofpH on the degree of inhibition of succinate dehydro-genase activity by Et2PC showed an inflection point around pH7.0 (Fig. 4), which is consistent with the pK. of histidyl imi-dazolium groups in proteins (21). Thus, the totality ofthe resultsappears to be consistent with the possibility that Et2PC modi-fication of succinate dehydrogenase involves an essential his-tidyl residue, apparently at the active site.The above results recalled earlier data on the effect ofpH on

succinate oxidase activity of SMP. It has been observed that,in contrast to the NADH and NADPH oxidase activities, the

Table 2. Reversal by hydroxylarnine of DEPC inhibition ofsuccinate dehydrogenase activity

% of control activityActivity remaining NH20H after NH20H

after Et2PC treatment, added, treatment% mM 30min 60min32 20 57 6534 24 42 6532 30 46 4534 115 35 46

Wavelength, nm

FIG. 3. Difference spectrum of Complex II in the presence ofEt2PC. Complex 1 (1 mg/ml) in 50 mM potassium phosphate, pH 6.0/0.07% lysolecithin was placed in the sample and reference cuvettes,and the base-line difference spectrum (trace 1) was recorded in the UVrange shown. Then, 10 p1 of 0.55M Et2PC in ethanol was added to thesample cuvette (1 ml) and an equal volume of ethanol was added to thereference cuvette. After 15 min the difference spectrum (trace 2) wasrecorded. At this point 20 A1 of 3 M hydroxylamine (neutralized) wasadded to each cuvette, and after 30 min the difference spectrum (trace3) was recorded.

succinate oxidase activity ofSMP decreased precipitously at pH< 7.0 (22). Therefore, we compared the effect of pH on thesuccinate oxidase and the succinate-fericyanide reductase ac-tivities of SMP and found that whereas between pH 7 and 9these activities were relatively constant, they both decreasedsharply atpH values below 7.0 (Fig. 5). Several possibilities maybe considered for this interesting pH profile, such as the effectofpH on the active conformation of the enzyme, on the rate ofelectron transfer by flavin and the iron-sulfur centers, etc.However, because the pK. of the imidazolium moiety of histi-dine in most proteins is between pH 5.5 and 7.0 (21) and theEt2PC inhibition studies had suggested the possible presenceofan essential histidyl residue in succinate dehydrogenase, pre-sumably at the active site, another interesting possibility for thepH profile ofsuccinate dehydrogenase activity presented itself.This possibility involves the participation ofthe histidyl residuein the mechanism of enzyme-substrate interaction and facili-

-1.0

-2. ~ ~ 0

-2.0' 6.0 7.0 8.0pH

FIG. 4. Effect of pH during incubation of purified succinate de-hydrogenase with 9.8 mM Et2PC on the pseudo-first-order inhibitionrate constant (k) of succinate:PMS/DCIP oxidoreductase activity.

Biochemistry: Vik and Hatefi

6752 Biochemistry: Vik and Hatefi

To >-

-CI,1

.C

CD CDCJ 0

00C.C CM

cmCoC3

0.0

-0.5

6 7 8pH

FIG. 5. Effect of pH on the succinate oxidase (e), succi-nate:ferricyanide oxidoreductase (A), and fumarate reductase (o) ac-tivities of SMP. Succinoxidase rates are at v,, with respect to suc-cinate concentration. Succinate:ferricyanide oxidoreductase activitywas assayed as described (2) in the presence of saturating amounts ofsuccinate and 3 mM potassium ferricyanide (higher ferricyanide con-centrations are inhibitory). It was necessary to use this assay for mea-surement of succinate dehydrogenase activity becausepH changes im-posed added effects whenPMS + Cl2IP were usedas electron acceptors.

tation ofsuccinate oxidation. In such a case, however, one wouldexpect the same histidyl residue to be involved also in the mech-anism of fumarate reduction and possibly expect a pH profileof fumarate reductase activity that would be a mirror image ofthe pH profile of succinate dehydrogenase activity.

These presumptions appeared to be correct for the pH profileof the fumarate reductase activity of SMP (Fig. 5). Thus, fu-marate reductase activity was highest and constant in the pHrange 5.5-7.0, where succinate oxidase activity diminished byabout 1 order of magnitude by decreasing the pH from 7.0 to5.5. In constrast, at pH 2 7.0, where succinate oxidase activitywas highest and constant, fumarate reductase activity dimin-ished as thepH was increased from pH 7.0 to 9.0. Similar resultswere obtained with the purified succinate dehydrogenase. Itmight be added that the dye chosen as reductant, phenosafra-nine, had a reduction potential (Em,7.0 = -252 mV) consider-ably more negative than that of the succinate/fumarate couple(Em.70 = +30 mV) and that, in the pH range used, this favorabledifference in the reduction potential of phenosafranine wasmaintained (Em = -184 mV at pH 5.45 and -302 mV at pH8.62) (23). Furthermore, the fumarate reductase activity in thepresence of reduced phenosafranine as electron donor was in-hibited 54%, 68%, and 80%, respectively, by 10 mM each ofsuccinate, malonate, and oxaloacetate and 84% by 90 ,uM of p-chloromercuriphenyl sulfonate, when the inhibitors wereadded to the reaction mixture. The fumarate reductase activityalso was inhibited by treatment of the enzyme with Et2PC, andsuccinate and fumarate protected the enzyme against Et2PCinhibition of fumarate reductase activity.

DISCUSSION

It has been shown that at pH 6.0, Et2PC inhibits succinate de-hydrogenase activity in SMP, Complex II, and purified succi-nate dehydrogenase. The inhibition was pseudo-first-orderwithrespect to incubation time, and the reaction order with respectto Et2PC concentration was close to unity, indicating that in-hibition resulted from modification of one protein residue byEt2PC per active unit of the enzyme. The pH profile of Et2PCinhibition, the UV spectrum ofthe Et2PC-treated enzyme, andthe partial reversal of inhibition by hydroxylamine suggested

that the essential group modified by Et2PC at pH 6.0 is mostlikely the imidazole moiety of a histidyl residue.t In addition,the findings that the substrates succinate and fumarate, the in-hibitors malonate and oxaloacetate, and the enzyme activatorsNaClO4 and NaBr all protected the enzyme against inhibitionby Et2PC suggested that the presumed essential histidyl resi-due is probably located at the active site. It has been shown thatsuccinate, fumarate, malonate, oxaloacetate, and Br- also pro-tect succinate dehydrogenase against inhibition by N-ethyl-maleimide (5). Thus, it appears that the essential SH group andthe putative essential histidyl residue might be located close toone another. In succinate dehydrogenase, FAD is covalentlybound to a histidyl residue ofthe larger subunit of the enzyme.The linkage involves the methyl group at position 8 ofthe isoal-loxazine ring and N-3 of the imidazole moiety of a histidyl res-idue. The amino acid on the amino end of the histidyl residueis serine, and on the carboxyl side of the histidyl residue, 21amino acids have been sequenced (24). This sequence does notinclude a histidyl (or a cysteinyl) residue, and whether the his-tidyl residue attached to FAD can still react with Et2PC at N-1 of the imidazole ring remains to be determined. We know,however, that in the experiment of Fig. 3, where the Et2PC-treated enzyme showed a spectral change in the UV region,there was no change in the visible spectrum of the enzyme inthe absorbance region of flavin. Whether attachment of anethoxyformyl group to the flavin-bound histidyl residue at N-1 might modify the visible absorption spectrum of the enzymealso remains to be investigated.The mirror-image pH profiles of succinate dehydrogenase

and fumarate reductase activities of Fig. 5 strongly suggest theparticipation of an unprotonated group in succinate oxidationand its protonated form in fumarate reduction. In the experi-ments of Fig. 5, the electron acceptor for succinate oxidationwas either the natural acceptor in the respiratory chain (suc-cinate oxidase assay) or the dye ferricyanide (succinate dehy-drogenase assay). The similar pH effects obtained indicate thatthe pH profiles were not related to the nature of the electronacceptor. The results shown in this paper for the pH profile offumarate reductase activity involved the use ofreduced pheno-safranine as the electron donor. The fact that the pH profile offumarate reductase activity also was not influenced by the effectof pH on phenosafranine oxidation is clear from our previousstudies on the effect ofpH on the oxidation ofcytochrome bWofComplex II by fumarate (25). These studies also showed a pHprofile identical to that shown in Fig. 5 for the fumarate re-ductase activity of SMP.

Whether the pH profiles of Fig. 5 are indicative of titrationof the putative essential histidyl residue of the enzyme remainsto be determined. However, the pK range suggested by thedata of Fig. 5 agrees with the pKa of the imidazole moiety ofhistidyl residues of most proteins and with the results of Fig.4. Ifwe assume on the basis of the above results that succinatedehydrogenase contains an essential histidyl residue at the ac-tive site, participating in succinate oxidation and fumarate re-duction, then a possible minimum mechanism might be asshown in Fig. 6. The unprotonated imidazole moiety of the es-sential histidyl residue would form a hydrogen bond with one(a) methylene hydrogen of succinate, thus labilizing the hy-drogen (actually H-) on the second (P) methylene group. Forfumarate reduction, the reduced FAD on the enzyme wouldpresumably donate a hydride ion to the f3 methenyl group. Insynchrony with this process, two of the double bond electrons

t Verification of Et2PC modification of histidyl residues by amino acidanalysis is not possible, because N-ethoxyformylimidazole is unstableunder acid and alkaline conditions (17).

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Natl. Acad. Sci. USA 78 (1981) 6753

0 - 0 0 - 0

C "Af-zyottwt C|FAD FADH2|

H-C'H -FC-H

9N \N....HC-H H-C HN+

0 - 0 0 - 0

FIG. 6. Proposed minimal mechanisms for succinate oxidation andfumarate reduction, involving a putative essential histidyl residue atthe enzyme active site. Shaded areas, segments of the succinate de-hydrogenase protein.

would move toward the a carbon, and the protonated, posi-tively charged form of the imidazole group would donate a pro-ton to the a methenyl group to stabilize the reduced structureof the substrate. It is also possible that there might be appro-priate electron-withdrawing groups on the enzyme in the vi-cinity of the y-carboxyl and/or ,3-methenyl groups in order tomake the ,B-methenyl group electrophilic and reducible. Weappreciate the fact that our proposed mechanism is, in princi-ple, more plausible for succinate oxidation than for fumaratereduction. However, it should be kept in mind that under op-timal conditions the rate of fumarate reduction as catalyzed bySMP or the purified succinate dehydrogenase is 1 order of mag-nitude smaller than the rate ofsuccinate oxidation, and this maybe a consequence of the relative activation energies involvedin these two reactions.

The authors thank Dr. Y. M. Galante for valuable discussions andpreparations of Complex II and succinate dehydrogenase and Mr. C.Munoz for the preparation of mitochondria. This work was supportedby U.S. Public Health Service Grant AM 08126.

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Biochemistry: Vik and Hatefi