TIIE JOURNAL OF DIOLOGICAL CFIEMISTRY Vol. 249, No. 9, Issne of May 10, BP. 2811-2815, 1974

I’rinled in U.S.A.

Evidence That apo-Reduced Nicotinamide Adenine Dinucleotide

Dehydrogenase and apo-Electron-transf erring

Flavoprotein from Peptostreptococcus

elsdenii Are Identical*

(Received for publication, November 12, 1973)

CAROLYN n. WHiTFIELD AND STEPHEN G. MAYHEW$

From the Department of Biological Chemistry, The University of Afichigan,, Ann Arbor, Michigan @lo/,

SUMMARY

Electron-transferring flavoprotein (ETF) and NADH de- hydrogenase, containing a novel orange flavin, have been highly purified from the strict anaerobe Pejtostrepfococcus

elsdenii. Both enzymes couple the oxidation of NADH to the reduction of dyes but only ETF couples the oxidation of NADH to the reduction of butyryl coenzyme A dehydrogenase (ETF activity). Disc gel electrophoresis in sodium dodecyl sulfate and urea, immunological studies, and amino acid analyses have provided strong evidence that the apoproteins of ETF and NADH dehydrogenase are very similar or identi- cal. The two enzyme preparations differ structurally in the proportions of their flavin chromophores, FAD and two novel modified flavins, 6-hydroxy-7,8 - dimethyl - 10 - (ribityl - 5’- ADP)-isoalloxazine and 7-methyl-8-hydroxy-lo-(ribityl-5’- ADP)-isoalloxazine (g-OH-FAD). Preparations with ETF activity contain FAD as the predominant prosthetic group and low amounts of the two modified flavins. NADH dehydro- genase preparations contain all three flavins, about 50% FAD and 50% modified flavins; there is a high proportion of g-OH-FAD which has an intense absorption band at 475 nm and imparts an orange color to the protein. Differences in catalytic activity, absorption spectrum, electrophoretic mo- bility, and isoelectric point of ETF and NADH dehydrogenase are most likely due to different proportions of the three flavins.

Two cwzpc preparations dkh osiclizc N:\I>Il IIRVC bern highly purifictl from tlic strict auaerol~r l’cptosfreptococczls

elsclenii. I~l(,~troll-trallsferriJlg flavoprotcin (1) aid NXl)II

tlcligtlrogeiJast: contaiiiiiig a novel orauge prosthetic group (2) couple the oxidation of NM111 to the rc>tluction oi” dyes

* This work was supported by (irant GRI 1llOG from the United States Public IIealth Service t)o I>r. Vincent i\I:~sscy.

$ Recipient of American Cancer Society Postdoctoral Fcllow- ship PF 670.

$ Present address, Laboratorium voor I~iochemic, I,andbou- whogeschool, J)e Driejen 11, Wageningcn, The Ncthcrlands.

(diaphorase activity). Only ETF’ (l-3) couplrs the oxidation

of NADH to the reduction of butyryl cocnzyrne ;2 tlehydro-

gcnase (ETF act,ivity). The major prosthetic group of ETF is FM1 (1); all preparations of ETF also contain variable amouiits of a grccii modified flavin, G-OI-I-FAD.2 I’rclimiiiary

studies which intlicatctl t,hat t,hc orange chromophore of NADII

tlehydrogenasc is a derivative of FAT) motlificd in the

isoallosa.zine ring (2) have been confirmed by ident,ification of

8-011.FAD as the orange prosthetic group (4).

Fro111 a comparison of the catalytic properties of purified

orange diaphorasc and the partially purified ETF of I~aldwin

ailtl Milligan (3)) it was iiiitially concluded that the two enzymes

are different (2). IIowever, our subsequent work with highly

purified ETF (1) has prompted a reappraisal of this view. It

was observed that all preparations of purified E’I’F contain

tracts of 8-OH-FM) and that, the procedure for the purification

of the orange diaphorase usually lcads to a mixture of the two

cnzgmcs. 111 this paper we prcscllt, evidence that NXDH de-

hytlrogcnase :IJKI ISTF are the same or very similar proteins

a11c1 differ structurally in their prosthetic group composition.

Some rcductiou properties of NADII dehydrogcnase are also

&scribed.

ISXI’ERIMESTAL PROClSJ)URE

Many of t,he materials and methods used in the present work have been described or referenced in the accompanying paper (1). Additional methods are described below. Disc gel electrophoresis at, pH 9.0 was performed according to the method of Ornstein and 1)nvis (5) and gels were stained with Coomassie blue (6).

1,,2?)Lu?/olog?/-Antibody to ETF and Ouchterlony plates were prepared by the general methods of Campbell cl (II. (7). New Zealand rabbi& were injected three times at weekly intervals subcutaneouslv in the back and foot nads with 0.5 ml of ISTF. 170 rg, in Freund’i adjuvant (Difco), 0.2E1 ml, and 0.07 hl NaCl. ‘One week after the last injection, blood was collected from the ear and the serum was separated and stored at -20”. Ouchterlony plates contained 0.1 M NaP i, pH 7.2, 0.85% NaCl, 1% sodium azide, and 1% Noble special agnr (IXfco).

1 The nbbrevixt,ions used are: 15TF, electroll-transferring flavo- protein; 8.OH-FAI), 7-methv-8-hvdroxv-lO-(ribitvl-5’-AI1P)-iso- alloxazine; 6.OH-FAl>, t.-hydr&7;8-di&thJ;l-10.(ribityl-5’- AI>P)-isoalloxazinc: 8.OH-FMN. 7-methvl-8.hvdroxv-lO-(ribitvl- 5’-phosphate)-isoitll;x2tzinc; SDS: sodium-dodecyl sulfate: ti

2 S. G. Mayhew, C. I). Whitficld, 8. Ghisla, and M. S. JBrns, Eur. J. Biochenf., in press.

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Prosthetic Groups-The prosthetic groups of NADH dehydro- genase were extracted with trichloroacetic acid and purified bv chromatography on DEAE-cellulose, a procedure which separates FAD. 8OH-FAD. and 6-OH-FAD.2, 3 The extinction coefficient of a chemically synthesized 7-methyl-%hydroxy-isoalloxazine model compound was determined from its weight and absorption spectrum to be 41,000 ~-1 cm-i at 472 nm (4).3 Since the extinc- tion coefficient of 8OH-FMN is expected to be the same as that of the model compound and the absorption of 8-OH-FAD is 10% less than 8-OH-FMN,3 the extinction coefficient of 8OH-FAD is 36,900 M-~ cm-i at 475 nm. The per cent of bound flavins was calculated from the spectra of NADH dehydrogenase, isolated FAD-ETF, 6-OH-FAD-apo-ETF, and 8.OH-FAD-apo-ETF using the following extinction coefficients: 12,500 M-I cm-1 at 450 nm for FAD-ETF (1) ; 20,300 M+ cm-i at 450 nm for 8-OH-FAD-ape-ETF; 2,210 M-I cm’; at’450 nm for 6-OH-FAD-apo-ETF.2 -

NADH Dehudroaenase Purification-NADH dehvdronenase was purified from Eellsgrown in iron-poor medium using DEAE-cellu- lose chromatography, ammonium sulfate fractionation, and Sephadex G-100 chromatography as described previously (2). In contrast with our early preparations made by this procedure which yielded the orange enzyme in high purity, our more recent preparations have given a mixture of this enzyme and ETF. Sometimes only traces of the orange diaphorase are present. These two proteins can be separated either by zone electrophoresis or by isoelectric focusing in Sephadex as described below.-

Thin Layer Bone Electrophoreais-Dry Sephadex G-100, 24 g, was suspended in 315 ml of 0.05 M KPi, pH 7.0, and the slurry was spread evenly over the surface of a glass plate (44 X 17 X 0.3 cm) bounded bv walls. 0.3 cm. of Plexielas. The elass nlate was nlaced on the metal cooling plate in a Savant Instrument high voltage electrophoresis apparatus. Water at 2” was circulated through the metal plate. Contact between the electrode vessels contain- ing 0.05 M KPi, pH 7.0, and the gel was made with Whatman No. 3MM paper strips covered with dialysis tubing. Protein, 25 mg, in 2.3 ml of 0.05 M KP,, pH 7.0, was pipetted in a line across the gel bed near the cathode end and the gel was covered with a layer of Parafilm. Electrophoresis was carried out for 24 hours at 300 volts and 90 ma with frequent changes of electrode buffer to main- tain the pH. At the completion of the run, colored and fluorescent sections of the gel were removed, suspended in 0.05 M KP ,, pH 7.0, poured into a column, and eluted with the same buffer.

Thin Layer Isoelectric Focusing-Isoelectric focusing was per- formed using the method of Radola (8) and the equipment de- scribed above. Dry Sephadex G-100 or G-75,7.5 g, was suspended in 100 ml of 1% amnholine. nH 3 to 6. from LKB nrodukter. The electrode vessels contained*O.2 M H&O4 at the anode and 0.25 M

NaOH at the cathode. The pH gradient was established by elec- trophoresis at 800 volts for 4 hours. After the current dropped from 25 ma to 4 ma, the protein in 5 X 10e3 M KP ,, pH 6 to 7, was applied to the gel near the cathode end in a region of the pH gradi- ent where the enzymes are stable. Isoelectric focusing was car- ried out for 24 hours or until there was no further movement of the colored bands. Colored or fluorescent bands were removed, suspended in 0.05 1\1 KP,, pH 7.0, applied to the top of a small Sephadex G-25 column, and eluted with the same buffer. This procedure separated ampholine from protein. Small sections of gel at 5-cm intervals were removed and suspended in 2 ml of water and the pH was measured to determine the pH gradient in the gel layer.

RESULTS

Purification of NADH Dehydrogenase-Recently the pro-

cedure for the purification of NADH dehydrogenase (2) re- sulted in a mixture of the orange diaphorase and ETF which could be separated by isoelectric focusing or zone elec- trophoresis. Thin layer isoelectric focusing of the protein ob- tained after the gel filtration step in the purification procedure separated two colored protein bands, an orange band correspond- ing to p1 5.4 and a yellow band at p1 4.9. A second yellow band was visible near the anode, but this was free FAD which probably dissociated from the protein-during the run. A similar separa-

3 S. Ghisla and S. G. Mayhew, manuscript in preparation

FIG. 1. Observed (--) and calculated (. . .) spectra of NADH dehydrogenase. The spectrum was calculated from the contribu- tions of 35yo 8-OH-FAD-apo-ETF(-. .-) 12% 6-OH-FAD-apo- ETF (- - -) ; and 53% isolated FAD-ETF (-.-).

tion was observed during thin layer zone electrophoresis at pH 7.0; an orange band migrated 16 to 17 cm from the origin near the cathode and a yellow band, 13 to 15 cm. In view of their p1 values and similar molecular weights, the relative electro- phoretic mobilities of the two proteins are surprising. The orange band had a spectrum similar to that previously reported for NADH dehydrogenase (2) (Fig. 1) and contained diaphorase activity. The yellow band had a spectrum similar to that for ETF purified by a different procedure (I) and exhibited ETF and diaphorase activities. The purified NADH dehydrogenase appeared about 90% pure by pH 9.0 polyacrylamide disc gel electrophoresis; one major band which contained NADH de- hydrogenase activity and two faint, enzymically inactive minor bands were observed.

Identity of apo-NADH Dehydrogenase and ape-ETP-SDS and urea electrophoresis, immunological studies, and amino acid analyses suggest that the apoprotein of NADH dehydro- genase is identical with apo-ETF. Like ETF, NADH dehy- drogenase is composed of two nonidentical subunits. The subunits of the orange diaphorase have the same molecular weights, 41,200 and 33,200, determined by SDS gel electro- phoresis, and the same charges, determined by urea gel elec- trophoresis, as the subunits of ETF (1). The distribution of protein between the orange diaphorase subunits is that ex- pected for a 1: 1 molar ratio. The sum of the subunit molecular weights differs somewhat from the native molecular weight, about 63,000, previously determined for NADH dehydrogenase (2), but it is in good agreement with more recent estimates of the native molecular weight, 72,000 to 75,000 of ETF (1). Fur- thermore, NADH dehydrogenase cross-reacts with antibody prepared against ETF. On Ouchterlony plates containing antibody to ETF in the center well and NADH dehydrogenase and ETF in the surrounding wells, the NADH dehydrogenase precipitin line intersects the ETF precipitin line without spurs (Fig. 2). This reaction of identity indicates that the two pro- teins have the same antigenic sites (7). It is unlikely that the antigenic reaction of NADH dehydrogenase is due to a small contamination with ETF since identical concentrations of the two proteins gave qualitatively the same antigenic reaction. Finally, the amino acid compositions of the orange diaphorase and ETF are very similar (Table I). The small differences are probably due to the contaminant which is known to be pres-

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FIG. 2. Reaction of ETF and NADH dehydrogenase with anti- body to ETF on an Ouchterlony plate. Center well: 0.04 ml of serum from rabbits immunized to purified ETF. Surrounding wells clockwise from cenler lop well: ETF, 5.5 rg; NASH dehydro- genase, 5.5 rg; ETF, 17 pg; NADH dehydrogenase, 17 pg; ETF, 55 pg; NADH dehydrogenase, 55 pg. There was no reaction with serum from rabbits not immunized to ETF.

ent in our preparations of NADH dehydrogenase. We con- clude from these observations that the apoproteins of ETF and NADH dehydrogenase are very similar and probably identical.

Prosthetic Groups and Absorption Spectrum-NADH de- hydrogenase and ETF differ in their prosthetic group com- position and thus in their absorption spectra. The major prosthetic group of ETF is FAD (I), but preparations of this protein also contain variable amounts of a novel green flavin that has recently been identified as 6-OH-FAD.2 Early in- dications that NADH dehydrogenase contains a modified flavin (2) have now been confirmed with the identification of 8-OH-FAD as the orange chromophore. The absorption spec- trum of 8-OH-FAD is shown in Fig. 3; there are maxima at 300 and 475 nm. However, this is not the only flavin in the orange protein. When the chromophores of NADH dehydrogenase are estracted from the apoprotein with trichloroacetic acid and then chromatographed on DEAE-cellulose, three colored bands, yellow, green, and orange are separated. These are respec- tively, FAD, 6-OH-FAD, and 8-OH-FAD.

Apo-ETF was titrated with purified 8-OH-FAD to deter- mine the spectrum of 8-OH-FAD-apo-ETF. The titration was followed by the difference spectrum between protein-bound and free 8-OH-FAD. The difference spectrum had masima at 520 and 320 nm and a minimum at 470 nm. The spectrum of 8-OH-FAD-apo-ETF has maxima at 483 and 312 nm and the extinction at 483 nm is 31,400 M-I cm-l (Fig. 3). Similar in-

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TABLE I

Amino acid composition of NADH dehydrogeltase and ETF

Amino acids

Aspartic acid. Threonine . Serine............... Glutamic acid. Proline. . Glycine. . Alanine. Valine............... Methionine. Isoleucine Leucine Tyrosine . Phenylalanine Lysine. Hi&dine . Arginine .

-

ETF= NADH dehydrogenaseb

38.8 38.4 15.3 16.4

6.3 8.1 37.3 38.7 17.6 16.0 32.2 32.4

41.1 38.0 29.0 28.9

6.1 5.3

28.6 27.2 28.1 27.9

6.5 6.9 9.6 10.4

27.3 26.1 4.5 2.9

10.5 10.5

0 Determined after 48 hours of hydrolysis and corrected for the amount of flavin which binds to isolated ETF and modified flavins (1).

b Determined after 48 hours of hydrolysis and normalized to ETF-stable amino acids; aspartic acid, glutamic acid, glycine, alanine, leucine, tyrosine. Cysteic acid and tryptophan were not determined.

350 450 Wavelength, nm

FIG. 3. Spectrum of free S-OH-FAD(- - -); corrected spectra of S-OH-FAD-apo-ETF (--) and NASH dehydrogenase (s..). The spectrum of 8-OH-FAD-apo-ETF was obtained from an early point in the titration of apo-ETF with &OH-FAD and was cor- rected for the small absorption of the apo-ETF. The spectrum of NADH dehydrogenase (Fig. 1) was corrected for the presence of FAD and 6-OH-FAD using the spectra of 6-OH-FAD-apo-ETF and isolated FAD-ETF (Fig. 1) and assuming that the peaks at 358 and 650 nm are due to 6-OH-FAD and the peak at 375 nm and 460 nm shoulder are due to FAD.

formation has been obtained for FAD-apo-ETF (1) and 6-011- FAD-apo-ETF.2 With these data, the complicated spectrum of the orange diaphorase can be accounted for in terms of the flavin components (Fig. 1). We conclude that the maxima in the NADH dehydrogenase spectrum at 480 and 320 nm and the shoulder at 510 nm are due to 8-OH-FAD; the maximum at, 375 nm and the shoulder at 460 nm are caused by FAD, and the masima at 650 aud 358 nm and the shoulder at 425 nm arise from 6-OH-FAD. The calculated spectrum of NADH dehy- drogenase with 35% 8-OH-FAD, 53% FAD, and 12% B-OH-

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FAD is similar to t.he spectrum observed with t.his preparation of NADH dehydrogenase (Fig. 1). Furthermore, the spectrum of NADH dehydrogenase corrected for the presence of 53 to 58% FAD and 12% g-OH-FAD is very similar to that of &OH-FAD apo-ETF (Fig. 3). These observations support the conclusion that apo-ETF and apo-NADH dehydrogenase are the same or very similar proteins.

Activity-As mentioned previously, ETF and NADH de- hydrogenase differ in their catalytic activities. Although both enzymes couple the osidation of NXDH to the reduction of 2,6-dichlorophenolindophenol (diaphorase activity), only ETF couples the oxidation of NADH to the reduction of butyryl-CoA 0.1 - dehydrogenase in t,he assay with crotonyl-CoA (ETF activity). From the end point in a titration of apo-ETF with flavin, the activity per nmole of bound flavin has been calculated. The 300 400 500 600 700

complex of 8.OH-FAD with apo-ETF has 0.77 unit of diaphorase WAVELENGTH, nm

activity per nmole of bound flavin compared with 2.46 units FIG. 4. Anaerobic titration of NADH dehydrogenase in 0.11 M

for FAD-apo-ETF. Like NADH dehyclrogenase and 6-OH- KPi, pH 7.0, with sodium dithionite at 20”. The CUTV~S are labeled FAD-ape-ETF, 8-OH-FAD-apo-ETF is inactive in t,he assay according to the molar ratio of dithionite to protein-bound flavin.

for ETF compared with 0.61 unit of ETF activity per nmole of Zjlsel, plot of absorption at 478 nm versus the molar ratio of di-

bound flavin for FAD-apo-ETF. The observed diaphorase thionite to protein-bound flavin at each step in the titration.

activity, 2.9 units per nmole of bound flavin, of NADH dehy- drogenase is close to the theoretical value, 2.7 units, calculated from its flavin composition and the known activities of FAD- apo-ETF, 8-OH-FAD-apo-ETF, and 6-OH-FAD -apo -ETF. Since NADH dehydrogenase preparations contain about 50% FAD, their lack of any ETF activity, even when purified at the p1-I optimum for ETF, is surprising. To test the effect of mix- tures of 8-OH-FAD and FAD 011 the catalytic activities of the protein, apo-ETF was incubated with FAD to fill 50% of the flavin sites and then incubated with sufficient 8-OH-FAD to fill the remaining sites. The diaphorase activity increased in both incubations as espected for binding of both flavins. However, the ETF activity present after the first incubation disappeared after the second incubation. III control experiments, when apo-ETF was incubated with 507, FAD for the same times, ETF activity did not disappear and when 8-OH-FAD was diluted into the assay mixture, the ETF activity of FAD-apo-ETF was not inhibited. Furthermore, when apo-ETF was first titrated with 8-OH-FAD to occupy about 75% of the flavin sites and then further titrated with FAD to occupy the remaining sites, the diaphorase activity increased throughout the two parts of the titration as expected for binding of both flavins but the final preparation was completely inactive in the assay for ETF. The same results were obtained when the reduction of the A450 of butyryl-CoA dehydrogenase was measured anaer- obically with catalytic FAD-apo-ETF or 8-OH-FAD-apo-ETF and excess NADH.

Reduction of NADH Dehydrogenase by Dithionite and NADH- When NADH dehydrogenase is titrated anaerobically with sodium dithionite the changes in absorption are not linear (Fig. 4). The peaks at 350 and 650 nm disappear early in the titra- tion, indicating that 6-OH-FAD is reduced before FAD and 8-OH-FAD. The dithionite required for complete reduction is about 1 molecule or 2 electrons per molecule of bound flavin. This suggests that as with ETF (1) there are no colorless oxi- dation-reduction active groups in the orange diaphorase. When NADH dehydrogenase is reduced anaerobically with NADH, the results are similar except that the protein is not quite com- pletely reduced by 1 mole of NADH per mole of bound flavin (Fig. 5). Towards the end of the titration a band of absorption with a broad maximum at 700 to 756 nm appears. A similar band is formed during the titration of ETF with NADH and is

....... \ \

01

t

.... ...... .!.?' ....

.... '\ \ \ i

I I I I 400

WIIJELE~GTH . nm 600 700

FIG. 5. Anaerobic titration of NADH dehydrogenase in 0.12 M

KPi, pH 7.0, with NADH at 20”. The curves are labeled according to the molar ratio of NADH to protein-bound flavin. Ztlset, plot of absorption at 478 nm versus the molar ratio of NADH to protein- bound flavin at each step in the titration.

possibly due to a charge transfer interaction of reduced flavin and NAD+ (1).

DISCUSSION

Disc gel electrophoresis in SDS and urea, immunological studies, and amino acid analyses have provided strong evidence that t.he apoproteins of P. elsdenii ETF and an orange protein, previously identified as an NADH dehydrogenase, are very similar or identical. These and other analyses suggest that the two enzyme preparations differ structurally in the proportions of three flavins, FAD, 6-OH-FAD, and 8-OH-FAD and that NADH dehydrogenase is a modified form of ETF. Preparations with ETF activity contain a low amount of modified flavins; preparations with only diaphorase activity contain about 59% modified flavins. Differences in the catalytic activity, ab- sorption spectrum, electrophoretic mobility, and isoelectric point of ETF and the orange diaphorase are probably due to different proportions of the three flavins.

All of our preparations of ETF contain the two modified

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flavins. The amounts of modified flavins have varied from 1 to 35cj, of the total flavin for &OH-FAD and from 5 to 300/ for 6.011-FAD. The variation does not correlate with the iron content of the medium, which is known to influence the production of flavodosin in P. elsdenii (9). The reason for this variation and the origin and function of the modified flavins is not yet known. The biological function of the modified flavins is not clear since they do not reduce butyryl-CoA dehydrogenase; however, they may function in transfer to an unknown electron acceptor or in control of ETF activity. It is also possible that the modified flavins arise during the purification of ETF when esposed to conditions not present in the cell, such as oxygen.

A striking feature of NADH dehgdrogenase is the complete lack of ETF activity in coupling the osidation of NADH to the reduction of butyryl-CoA dehydrogenase even though it con tams a substantial amount of FAD. Considering this observa- tioii and that there are two flavin sites per molecule of apo-ETF

(l), and that NADH dchydrogennse which contains about 50% F,11) and 507, modified flavins is separated from FAD-ETF by isoelectric focusing or clectrophorcsis, we conclude that most NADH dehydrogenasc molecules contain 1 molecule of FAD and 1 molecule of 8-OH-FAD or R-OH-FAD. This suggests that modified flavins interfere with the ETF activity of FAD in the same protein molecule or that both flavin sites are needed for ETF activity. It seems unlikely that both flavin sites are needed for ETF activity because ETF activity per nmole of bound FhD is constant during the titration of apo-ETF with FAD (1) and most protein molecules have bound only 1 mole- cule of FAD early in the titration. The possibility that protein molecules simultancouslg bind 2 molecules of flavin is doubtful because of the absence or disappearance of ETF activity with the following mistures of FAD, K-OH-FAD, and apo-ETF. ETF activity is completely absent in preparations of apo-ETF where 757, of the flavin sites arc first filled with &OH-FAD and then the remaining sites are filled with FAD. ETF activity, initially present after incubation of apo-ETF with FAD to fill

50% of the flavin sites, disappears after subsequent incubation with 8-OH-FAD to fill the remaining sites. Redistribution of flavins in these experiments is unlikely since the rate at which FAD is released from the FAD-apo-ETF complex, k,rr, is very slow, approsimately IO-* min-*.4 These results support our conclusion that only one flavin site is necessary for ETF ac- tivity and that modified flavins prevent ETF activity of FAD in the same protein molecule possibly by inducing a conforma- tional change in the protein. Such a conformational change and the weakly acidic hydroxyl groups of the modified flavins could esplain the fractionation of NADH dehydrogenase and ETF by isoelectric focusing and electrophoresis.

Acknowledgments-We thank Dr. Vincent Massey for provid- ing facilities and Drs. Sandro Ghisla, V. Massey, and Charles Williams, Jr. for their valuable discussions.

REFERENCES

1. WHITFIELD, C. I>., AND MAYHE\\‘, S. G. (1974) J. Biol. Chem. 249, 2801-2810

2. MAYHEM, S. G., AND MASSEY, V. (1971) Biochim. Biophys. Acfa 236, 303-310

3. BALDVTN, R. L., AND MILLIGAN, L. P. (1964) Biochim. Biophys. Actw 92, 421-432

4. GHISLA, S., AND MAYHEW, S. G. (1973) J. Biol. Chem. 248, 6568-6570

5. ORNSTEIN, L., AND DAVIS, B. J. (1964) Ann. N. Y. Acad. hi. 121, 321-349

6. Wsuira, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406- 4412

7. CAMPUELL, I). II.. GARVEY, J. S., CREMER. N. E., AND Suss- DOIZF, D: H. (1964) in Melhods in Zmmurblogy, pp. 143-149, 97-99. W. A. Benjamin. New York

8. RADOL.;, B. J. (1666) Biolhim. Biophys. Acta 194, 335-338 9. M.IYHER, S. G., AND MASSEY, V. (1969) J. Biol. Chem. 244,

794-802

4 The approximate k,,rr was calculated from the dissociation constant for FAD-ano-ETF. & = k,ct:k,, = 2 X ~O+M (1). and the rate of binding-of FAC tdapo-&‘?F;-k,, = i03 ~-1 rhii-1 at 10” (Whitfield, unpublished kinetic data).

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Carolyn D. Whitfield and Stephen G. MayhewIdentical

ArePeptostreptococcus elsdeniiand apo-Electron-transferring Flavoprotein from Evidence That apo-Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase

1974, 249:2811-2815.J. Biol. Chem. 

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