Isolation and characterization of a ferredoxin from Mycobacterium smegmatis takeo

Biochimica et Biophysica Acta, 743 (1983) 91-97 Elsevier Biomedical Press

91

BBA 31517

I S O L A T I O N AND CHARACTERIZATION OF A FERREDOXIN F R O M MYCOBA CTERIUM SMEGMA TIS TAKEO TAKEO IMAI a TAKASHI MATSUMOTO a.., SUMIO OHTA a.**, DAIJIRO OHMORI b, KOJI SUZUKI b, JUNZO TANAKA c MASAYUKI TSUKIOKA c and JIRO TOBARI a

a Department of Chemistry, Rikkyo (St. Paul's) University, Nishi-lkebukuro, Toshimaku, Tokyo 171, b Department of Chemistry, School of Medicine, Juntendo University, Narashino, Chiba 275, and c National Institute for Research in Inorganic Materials, Namiki 1-1, Sakura-mura, Niihari-gun, Ibaraki 305 (Japan) (Received September 21st, 1982)

Key words: Ferredoxin; Fe-S content; Absorption," ESR; (M. smegmatis)

A soluble ferredoxin was isolated in a crystalline form from Mycobacterium smegmatis Takeo. (This species has been identified as Mycobacterium avium strain Takeo in some previous papers. See Kusunose, M., et al. (1976) Arch. Microbioi., 108, 65-73, for the rationale for this new name.) The molecular weight was calculated to be 12035 from the amino acid sequence (Hase, T., et al. (1979) FEBS Lett., 103, 224-228). It contained 5.7 tool non-heine iron, 5.9 mol acid-labile sulfur and 8 mol cysteine residues per 12 035 g. The ferredoxin exhibited absorption maxima at 280 and 406 nm with shoulders around 330 and 450 nm, and the absorbance ratio ( A 4 0 5 / / A 2 8 0 ) w a s 0.61. The absorbance in the visible region was partially reduced by the addition of sodium dithionite, but was not affected by the addition of potassium ferricyanide. The two midpoint oxidation-reduction potentials at pH 7.0 were determined potentiometrically to be - 15 and - 4 3 5 mV. The EPR spectra in the isolated state exhibited an almost isotropic signal at g 2.00, while in the dithionite-reduced state the signal was at g 2.015. Though its physiological role in the cell is unknown, the ferredoxin served as an electron mediator for cytochrome c reduction by N A D P H in the presence of spinach ferredoxin-NADP + reductase. From the data described above, M. smegmatis ferredoxin would seem to be a two Fe-S cluster containing ferredoxin, and at least one of the two Fe-S dusters is thought to be of the 3Fe-3S type.

Introduction Two kinds of ferredoxin were isolated and characterized from Mycobacterium flavum, a nitrogenfixing bacterium [1,2]. These are the first ferredo-

* Present address: Faculty of Domestic Science, Showa Women's University, Taishido, Setagaya-ku, Tokyo 154, Japan. ** Present address: Toho Laboratories Co. Ltd., Higashine-shi, Yamagata 999-37, Japan. Abbreviations: PCMB, p-chloromercuribenzoate; SDS, sodium dodecyl sulfate; Era, midpoint oxidation-reduction potential; Taps, 3 -([2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]-amino)- 1propanesulfonic acid. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

xins isolated from the genus Mycobacteriaceae. In M. smegmatis Takeo, an aerobic, non-nitrogen-fixing bacterium, we found that relatively large amounts of a ferredoxin existed in the soluble fraction. After purification, the ferredoxin's amino acid sequence was determined and a unique distribution of cysteine residues in the sequence was reported [3]. This cysteine distribution is similar to that of Thermus thermophilus ferredoxin [4], Azotobacter vinelandii ferredoxin I [5] and Pseudomonas ovalis ferredoxin [6]. Recently X-ray crystallographic analysis [7] and M6ssbauer spectroscopy [8] has established that A. vinelandii ferredoxin I contains a novel 3Fe-3S

92 cluster in addition to a 4Fe-4S cluster. The 3Fe-3S cluster is believed to be present in some iron-sulfur proteins, based on various physicochemical data (e.g., Ref. 9). In the case of M. smegmatis ferredoxin, the presence of a 3Fe-3S cluster was suggested by the similarity of its amino acid sequence to that of A. vinelandii ferredoxin I [7]. The present communication describes the isolation and crystallization of a ferredoxin from M. smegmatis Takeo grown on a nutrient broth. The physicochemical properties and amino acid sequence thus derived are compared with those of various iron-sulfur proteins and the types of Fe-S clusters of M. smegmatis ferredoxin are posited. Materials and Methods

Materials DEAE-cellulose (Whatman DE-32) was obtained from Whatman Ltd. Sephadex G-100 superfine and Sepharose 4B were purchased from Pharmacia Fine Chemicals. All other chemicals were reagent grade and were purchased from commercial sources. Spinach ferredoxin and ferredoxin-NADP ÷ reductase were prepared as reported previously [10] according to the methods of Buchanan and Arnon [11] and Shin [12], respectively. Growth of bacterium M. smegmatis Takeo was grown on the glycerol-peptone-bouillon medium at 37°C for 6 days as described previously [13]. Methods Absorption spectra were measured by a Union SM-401 recording spectrophotometer at 20 + 1°C. Protein concentration was determined by five methods: biuret [14], rnicrobiuret [15], FolinCiocalteu reagent [16], dry weight [17] and amino acid analyses [18], using bovine serum albumin as a standard. In the experiments for amino acid analysis, aspartic acid, glycine, alanine, lysine and valine were used as standard amino acids after 72 h hydrolysis to calculate the protein concentration. Iron [19] and acid-labile sulfur [20] were determined colorimetrically. Molecular weight was calculated from amino acid composition [3]. SDS-

polyacrylamide gel electrophoresis was performed according to Ref. 21. Polyacrylamide gel electrophoresis at pH 8.9 was performed according to the method of Davis [22]. The oxidation-reduction potential of the ferredoxin was measured according to the method of Dutton [23]. The potentiometric titration was performed in an anaerobic glass vessel in an argon atmosphere in 0.1 M Taps at pH 8.5 with a TOADEMPA HM-5A pH meter fitted with a reference electrode (silver/silver chloride-platinum electrode, TOA-DEMPA PS 115C). The reduction of the ferredoxin by freshly prepared 30 mM sodium dithionite was monitored by an absorbance change at 420 nm with a Hitachi 124 spectrophotometer. To obtain rapid equilibrium between the ferredoxin and the reference electrode, the following 5 # M mediators were used: methylene blue ( E m = + 11 mV), 2-hydroxy- 1,4-naphthoquinone ( E m = - 1 5 2 mV), sodium anthraquinone-2-sulfonate (Em=-225 mV), phenosafranine ( E r a = - 2 5 2 mV), benzyl viologen ( E m = - 350 mV) and methyl viologen ( E m = - 445 mV). The absorbance change at 420 nm was corrected by subtraction of the absorbance attributed to the mediators alone at this wavelength. The data were fitted to Nernst's equation by computer simulation. EPR measurements were performed on an Xband JM-ME-3 type spectrometer (Japan Electron Optics Laboratory Co. Ltd.). The cavity mode was TEl02. The magnetic field was calibrated by a proton-resonance probe. The sample was introduced into a quartz tube of 2-mm inside diameter and first cooled with liquid nitrogen, then with liquid helium. The spectrum was recorded at liquid helium temperature (4.2 K). The stability of the ferredoxin at various pH was measured at 20°C as follows: the ferredoxin (6.9 m g / m l ) was diluted into the buffer (50 mM) to give a final concentration of 69 # g / m l . The buffers used were pH 3 (glycine-HC1), pH 4 - 6 (succinate-NaOH), pH 6.8 (sodium phosphate), pH 8-9 (Tris-HCl) and pH 9-11 (glycine-NaOH). The thermal stability of the ferredoxin was also measured in 50 mM Tris-HC1 buffer, pH 8.0, at the concentration of 69/~g/ml by incubating it for 10 min at various temperatures (30-70°C). Both pH and thermal stability were monitored by the decreases of the absorption at 406 nm.

93

Isolation of a ferredoxin All purification procedures were carried out at 0 - 7 ° C unless otherwise indicated. The cells were disrupted by sonic oscillation instead of the grinding with quartz sands reported earlier [24]. To the cell-free extract, solid ammonium sulfate was added. The precipitates formed by the addition of solid ammonium sulfate (35-80% saturation) were collected and suspended in a minimal volume of 50 m M Tris-HC1 buffer, p H 8.0 (hereinafter referred to as the buffer). This suspension was dialysed against 3 liters of the buffer overnight. Ferredoxin was adsorbed on DEAE-cellulose (DE32) by a batch-wise procedure. The DEAE-cellulose after adsorbing the proteins was washed with 700 ml of the buffer containing 0.15 M NaC1 and eluted with the buffer containing 0.7 M NaC1 on a Buchner funnel. To the eluted solution were added four volumes of the buffer. Then the diluted solution was applied on a DEAE-cellulose column (3.5 x 45.0 cm) previously equilibrated with the buffer. The ferredoxin absorbed on top of the DEAE-cellulose column was eluted with 3 liters of the buffer containing 0.29 M NaC1. To the brown-colored eluate, solid ammonium sulfate was added to give a final concentration of 3.5 M. It was then applied on a Sepharose 4B column (2.5 x 30 cm) equilibrated with the buffer containing 3.5 M ammonium sulfate. The column was washed with the same buffer (800 ml) and eluted with the buffer containing 2.9 M ammonium sulfate. The brown-colored eluates were collected, then concentrated with a m m o n i u m sulfate precipitation. The precipitate was dissolved in a minimal volume of the buffer, and passed through a Sephadex G-100 column (3.5 x 50 cm) equilibrated with the buffer containing 0.5 M NaC1. The eluates from the Sephadex column with an absorbance ratio (406 n m / 2 8 0 nm) of 0.61 were collected. Approximately 20 mg of the purified protein were obtained. Results

Purity Upon subjection to polyacrylamide SDS-polyacrylamide gel electrophoreses band was observed in each system and the purified protein was found to be neous.

gel and only one therefore homoge-

Crystallization To the purified ferredoxin solution concentrated to about 50 mg per ml was added finely divided ammonium sulfate until the solution became slightly turbid. A very small aliquot of water was then added until the solution became clear. After standing at 5°C for a few days, the ferredoxin crystallized as brown needles. Molecular weight From the amino acid sequence of the ferredoxin [3], the molecular weight was calculated to be 12035, assuming that it contained 6 iron and 6 sulfur. The molecular weight of 12035 is used throughout this work. Content of non-heme iron and acid-labile sulfur Contents of non-heme iron and acid-labile sulfur of the ferredoxin were measured as described in Materials and Methods. Non-heme iron contents shown in Table I indicate that M. smegmatis ferredoxin contains on average 5.7 atoms of non-heme iron per mole of the ferredoxin. The content of acid-labile sulfur was found to be 5.9 atoms per mole of the ferredoxin when the protein concentration was determined by the microbiuret method. pH and thermal stabilities The ferredoxin was fairly stable at p H 4.8-9.6 at 20°C for 20 h incubation monitored by an absorbance decrease at 406 nm. The absorbance decrease was within 5% at this p H range, but it

TABLE I DETERMINATION OF IRON CONTENT OF M. SMEGMATIS FERREDOXIN The protein concentrations were determined by five methods and iron content was determined colorimetrically,as described in Materials and Methods. Method

Iron content (atom/mol ferredoxin)

Microbiuret Biuret Folin-Cioealteu Amino acid analysis Dry weight

5.74 4-0.90 4.93 4-0.28 5.35 + 0.16 5.66 + 0.56 6.824-0.21

94 was 54% and 87% at pH 3.9 and 10.0, respectively. On the other hand, an absorbance decrease was observed by increasing the incubation temperature. The thermal stability of the ferredoxin decreased rapidly at 55°C. The pH and thermal stability of M. smegmatis ferredoxin was rather low compared to that of T. thermophilus ferredoxin, which was stable at pH 3.5-9.0 on overnight incubation at room temperature and was also stable at 67°C for 45 min [4].

Absorption spectra The absorption spectra of M. smegmatis ferredoxin were measured in the isolated and dithionite-reduced state. As shown in Fig. 1, the isolated ferredoxin has absorption maxima at 280 and 406 nm with shoulders in the region of 310-330 and 450-470 nm. This spectrum resembles those of the bacterial-type ferredoxins [25]. The absorbance ratio (406 n m / 2 8 0 nm) of the purified ferredoxin was 0.61 and the millimolar extinction coefficient at 406 nm was calculated to be 26.0 mM - l . c m - l , using the value of protein concentration determined by the microbiuret method. The visible absorption spectrum was reduced to 15-20% at pH 8.0 by the addition of solid sodium dithionite. Its reduction rate was slow: about 20 min were required for the maximal reduction. The reduced spectrum was markedly

reversed to the isolated state level when oxidized with air. In contrast, the absorption spectrum was not changed by the addition of potassium ferricyanide (3 and 10 mol per mol ferredoxin).

Oxidation-reduction potentials (Em) The midpoint oxidation-reduction potential of the ferredoxin was determined potentiometrically, monitored by absorbance changes at 420 nm. The result is shown in Fig. 2. The curve in the figure was obtained from Nernst's equation by computer simulation. The two E m values of the ferredoxin obtained were - 1 5 mV with n = 0.6, and - 4 3 5 mV with n = 0.8, respectively. The result suggests that the M. smegmatis ferredoxin contains at least two redox centers, whose potentials are approximately 400 mV apart, and that it might conduct one electron oxidation-reduction at its t w o E m values. (Em)

EPR spectra EPR spectra of the ferredoxin were measured at liquid helium temperature (4.2 K). The isolated ferredoxin exhibited a nearly isotropic signal at g 2.00. (Fig. 3A) This signal is similar to that of other bacterial ferredoxins in the oxidized state, such as T. thermophilus ferredoxin [4], D. gigas ferredoxin I and II [26] and A. vinelandii ferredo-

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REDOX POTENTIAL (mV)

i

500

O.OI

600

(nm)

Fig. 1. Absorption spectra of M. smegmatis ferredoxin. The spectra were recorded in 50 m M Tris-HCl, pH 8.0, containing 0.5 M NaCI, at 2 0 + I°C. The protein concentration was 10.7 ~M. Solid line, isolated state; dashed line, reduced state with excess solid sodium dithionite.

Fig. 2. Midpoint oxidation-reduction potentials of M. s m e g m a tis ferredoxin. The ferredoxin was dissolved in 0.1 M Taps, pH 8.5, at a concentration of 9.7 /~M. The measurements were carried out five times and all the data obtained were plotted on the same figure, while the control experiments (minus ferredoxin with dyes) were performed four times and the results subtracted from the observed values.

95

2.02

Reaction with P C M B T h e reaction r a t e of the ferredoxin with P C M B was examined. Fig. 4 shows the relatively slow r e a c t i o n rate of M. smegmatis ferredoxin with P C M B , c o m p a r e d with that of spinach or clostridial ferredoxin, a n d its nearly equal reaction rate with that of A. vinelandii ferredoxin I [29]: a b o u t 40 min were required for 50% of the m a x i m a l reaction, d e t e r m i n e d f r o m the a b s o r b a n c e increase at 250 n m after 30 h. In the presence of 6 M g u a n i d i n e h y d r o c h l o r i d e , a b o u t 80% reacted within 5 min. A t the e n d of the r e a c t i o n (after 30 h), a p p r o x . 24.8 mol P C M B h a d reacted with 1 mol. ferredoxin.

A

I 1.99 2.02

B

Cytochrome c reduction T h e physiological function of the ferredoxin is obscure, yet it has the ability to replace spinach ferredoxin in the r e d u c t i o n of c y t o c h r o m e c with spinach ferredoxin-NADP ÷ reductase a n d N A D P H . Its ability to replace was a b o u t 70% that o f spinach ferredoxin. (Fig. 5)

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MAGNETIC FIELD (Gouss) Fig. 3. EPR spectra of M. smegmatis ferredoxin. A, as isolated; B, reduced with excess sodium dithionite. The ferredoxin was dissolved in 50 mM Tris-HCl, pH 8.8, containing 0.5 M NaCI at a concentration of 34 mg/ml. Spectra were recorded as follows: A, microwave frequency, 9.186 GHz; microwave power, 10 mW; modulation amplitude, 4G; temperature, 4.2 K. B, microwave frequency, 9.168 GHz; microwave power, 10 mW; modulation amplitude, 2.5 G; temperature. 4.2 K.

I

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B

TIME

(rain)

.

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0.3

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xin I [27]. This signal is thought to be derived f r o m the oxidized [3Fe-3S] cluster [9]. U p o n r e d u c t i o n with excess a m o u n t s of s o d i u m dithionite, the g 2.00 signal d i s a p p e a r e d and a n o t h e r signal app e a r e d at g 2.015. (Fig. 3B) This r e d u c e d s p e c t r u m is rather different from those of m a n y o t h e r red u c e d b a c t e r i a l ferredoxins r e p o r t e d so far; i.e., T. thermophilus ferrdoxin [4] a n d D. gigas ferredoxins I a n d II [26]. W h e n p o t a s s i u m ferricyanide was a d d e d to the isolated ferredoxin, n o a p p r e c i a b l e increase of E P R signal intensity was observed. This result is different f r o m those of A. vinelandii ferredoxin I [27], R. rubrum ferredoxin IV [28] a n d M. flavum ferredoxin I [2].

I0

20

TIME (mini

30

0

I

2

3

4

Fig. 4. Reaction of M. smegmatis ferredoxin with PCMB. The reaction mixture (2 ml) contained the following: (A) 4.2 nmol ferredoxin and 193 nmol PCMB in 10 mM potassium phosphate, pH 7.0. (B) The same as in (A) except that 6 M guanidine hydrochloride was added. Reactions were carried out aerobically at 20+ I°C. Fig. 5. Cytochrome c reduction activity of M. smegmatis ferredoxin. The reaction mixture (1 ml) contained 10 #mol TrisHC1, pH 7.8, 0.25 Fmol horse heart cytochrome c, 20 vmol NADPH, 4 #g spinach ferredoxin-NADP + reductase and either 20.8 nmol M. smegmatis ferredoxin or 20.8 nmol spinach ferredoxin. The reduction of cytochrome c was measured at 550 nm at 20+ I°C. A, spinach ferredoxin; B, Mr. smegmatis ferredoxin; C, without ferredoxin.

96 Discussion

A ferredoxin was purified homogeneously and characterized from the soluble fraction of M. smegmatis Takeo grown aerobically on nutrient broth medium. It is probable that M. smegmatis ferredoxin contains two Fe-S clusters, at least one of which is of the [3Fe-3S] type. It thus resembles those ferredoxins that have two redox centers and function at different E m values, such as A. vinelandii ferredoxin I [27], R. rubrum ferredoxin IV [28], M. flavum ferredoxin I [2], D. gigas ferredoxin I [26] and T. thermophilus ferredoxin [4]. In order to clarify the type of the Fe-S clusters of M. smegmatis ferredoxin, some of its properties - - EPR spectra, effects of potassium ferricyanide addition and amino acid sequence - - were compared with those of other iron-sulfur proteins. The EPR spectrum of the isolated-state M. smegmatis ferredoxin, with a nearly isotropic signal at g 2.00, was quite similar to that of the isolated-state A. vinelandii ferredoxin I [27], beef heart mitochondrial aconitase [30], D. gigas ferredoxin II [26] and T. thermophilus ferredoxin [4], all of which contain [3Fe-3S] type cluster(s). We can therefore assume that the EPR signal at g 2.00 originates from the oxidized [3Fe-3S] cluster of M. smegmatis ferredoxin. On the other hand, the dithionite-reduced M. smegmatis feredoxin exhibited a signal at g 2.015. This signal differed from the g 1.94 centered signal which is derived from the [2Fe2S] 1÷ or [4Fe-4S] 1÷ cluster, but rather resembled the signal at g 2.01 observed in the reduced A. vinelandii ferredoxin I [21]. At the present time, however, it is not clear from what kind of Fe-S cluster the g 2.015 signal of the reduced M. smegmatis ferredoxin originated. When potassium ferricyanide was added to A. vinelandii ferredoxin I, both the absorption in the visible region and the EPR signal at g 2.01 increased [27]. These phenomena were also observed in R. rubrum ferredoxin IV [28] and M. flavum ferredoxin I [2]. As reported in C. pasteurianum 214Fe-4S] ferredoxin, an increase of the EPR signal intensity with the addition of potassum ferricyanide can be attributed to a conversion of the [4Fe-4S] cluster to a [3Fe-3S] cluster [9]. Neither the increase in the EPR signal nor the absorption in the visible region was observed when potassium

ferricyanide was added to M. smegmatis ferredoxin. These results suggest that the [4Fe-4S] cluster that can be converted to the [3Fe-3S] cluster by potassium ferricyanide was not present in M. smegmatis ferredoxin. The presence of a [4Fe-4S] cluster in M. smegmatis ferredoxin has been suggested by the same distribution of the four cysteine residues, at positions 24, 39, 42 and 45, as that of A. vinelandii ferredoxin I, in which the [4Fe-4S] cluster is ligated by these cysteine residues. However, results confirming the existence of the [4Fe-4S] cluster in M. smegmatis ferredoxin have not been obtained. The precise type of the two Fe-S clusters and the ligands of these clusters in M. smegmatis ferredoxin remains open. It is of great interest to clarify these problems in connection with the unusual EPR signal in the reduced state of the unique distribution of cysteine residues compared with those of A. vinelandii ferredoxin I [5] and T. thermophilus ferredoxin [4]. These problems will be elucidated using different physical measurements, such as X-ray crystallopgraphic analysis. Acknowledgements

The authors wish to thank Dr. T. Kimura, Wayne State University, for his helpful discussions and also Drs. I. Muramatsu and Y. Motogi of Rikkyo university for amino acid analyses. We would also like to express our sincere gratitude to Miss. M. Fukuda for typing the manuscript, and to Mr. S. Takayama for chemical analysis of the ferredoxin. References 1 Bothe, H. and Yates, M.G. (1976) Arch. Microbiol. 107, 25-31 2 Yates, M.G., O'Donnell, M.J., Lowe, D.J. and Bothe, H. (1978) Eur. J. Biochem. 85, 201-299 3 Hase, T., Wakabayashi, S., Matsubara, H., Imai, T., Matsumoto, T. and Tobari, J. (1979) FEBS Lett. 103, 224-228 4 0 h n i s h i , T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K. and Oshima, T. (1980) J. Biol. Chem. 255, 345-348 5 Howard, J.B., Lorsbach, T. and Que, L. (1976) Biochem. Biophys. Res. Commun. 70, 582-588 6 Hase, T., Wakabayashi, S., Matsubara, H., Ohmori, D. and Suzuki, K. (1978) FEBS Lett. 91,315-319 7 Ghosh, D., Furey, W., Jr., O'Donnell, S. and Stout, C.D. (1981) J. Biol. Chem. 256, 4185-4192

97 8 Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H. and Miinck, E. (1980) J. Biol. Chem. 255, 1793-1796 9 Johnson, M.K., Spiro, T.G. and Mortenson, L.E. (1982) J. Biol. Chem. 257, 2447-2452 l0 Matsumoto, T., Tobari, J., Suzuki, K., Kimura, T. and Tchen, T.T. (1976) J. Biochem. 79, 937-943 II Buchanan, B.B. and Arnon, D.I. (1971) Methods Enzymol. 23A, 413-439 12 Shin, M. (1971) Methods Enzymol. 23A, 440-446 13 Kimura, T. and Tobari, J. (1963) Biochim. Biophys. Acta 73, 399-405 14 Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 117, 751-756 15 Itzhaki, R.F. and Gill, D.M. (1964) Anal. Biochem. 9, 401-410 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 17 Hong, J.-S. and Rabinowitz, J.C. (1970) J. Biol. Chem. 245, 4982-4987 18 Moore, S. and Stein, W.H. (1963) Methods Enzymol. 6, 819-831 19 Massey, V. (1957) J. Biol. Chem. 229, 763-770 20 King, T.E. and Morris, R.O. (1967) Methods Enzymol. 10, 634-637

21 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 22 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 23 Dutton, P.L. (1971) Biochim. Biophys. Acta 226, 63-80 24 Imai, T. (1978) Biochim. Biophys. Acta 523, 37-46 25 Yoch, D.C. and Carithers, R.P. (1979) Microbiol. Rev. 43, 384-421 26 Cammack, R., Rao, K.K., Hall, D.O., Moura, J.J.G., Xavier, A.V., Bruschi, M., LeGall, J., Deville, A. and Gayda, J.-P. (1977) Biochim. Biophys. Acta 490, 311-321 27 Sweeney, W.V., Rabinowitz, J.C. and Yoch, D.C. (1975) J. Biol. Chem. 250, 7842-7847 28 Yoch, D.C., Carithers, R.P. and Arnon, D.I. (1977) J. Biol. Chem. 252, 7435-7460 29 Yoch, D.C. and Arnon, D.I. (1972) J. Biol. Chem. 247, 4514-4520 30 Ruzicka, F.J. and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517 31 Shethna, Y.I. (1970) Biochim. Biophys. Acta 205, 58-62 32 Moura, J.J.G., Moura, I., Kent, T.A., Lipcomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V. and Miinck, E. (1982) J. Biol. Chem. 257, 6259-6267