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THE JOURNAL OF Bro~oo~ca~ CHEMISTRY Vol. 246, No. 3, Issue of February 10, pp. 643-653, 1971 Printed in U.S.A. The Oxygen Sensitivity of Spinach Ferredoxin and Other Iron-Sulfur Proteins THE FORMATION OF PROTEIN-BOUND SULFUR-ZERO* (Received for publication, June 19, 1970) DAVID PETERING,~ JAMES A. FEE,§ AND GRAHAM PALMERS From the Department of Biological Chemistry, Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Michigan 48105 SUMMARY In the presence of urea and molecular oxygen, spinach ferredoxin is rapidly denatured. The product of this reac- tion contains substantial amounts of sulfur in the oxidation state of zero covalently bound to the protein. The origin of the sulfur-zero was shown to be the labile sulfide of the native protein. The S(0) could be released from the pro- tein as S- with a strong reducing agent such as dithio- threitol or as S(0) with cyanide or sulfite anions. Sedi- mentation analysis showed the protein to be polymeric, existing as a dimer. Ferricyanide could replace oxygen as a source of oxidizing equivalents. With this oxidant the stoichiometry of the reac- tion was found to be 9 moles per mole of ferredoxin. This has been explained by the oxidation of 5 RSH to S/2 RSSR and 2 S2- to 2 S(0). It is proposed that the S(0) is bound to the protein as a trisutide (RSSSR). The ferricyanide titer of a number of iron-sulfur proteins has been determined and correlated with the S(0) content of the urea-ferricyanide denatured proteins. The oxidation stoichiometries are consistent with a generalization of the above formulation. Thus 8 to 10 moles of ferricyanide are consumed by the two-iron proteins and approximately 30 by the bacterial ferredoxins. With the two-iron proteins, 60 to 80% of the labile sulfide was found as bound S(0) while with bacterial ferredoxins about 50% of the labile sulfide was detected as bound S(0). In the case of putidaredoxin it was found that the same amount of S(0) was bound when the protein was air-de- natured in the absence of urea and mercaptoethanol as when the protein was denatured in aerobic urea solution. It is proposed that the oxygen sensitivity of many non-heme iron labile-sulfide proteins finds explanation in the oxidation of their mercaptide-sulfide system. * This work was supported by National Institutes of Health Research Grant GM 12176. $ National Science Foundation Predoctoral Fellow. Present address, Department of Chemistry, Northwestern University, Evanston, Illinois 60201. $ National Institute of Health Postdoctoral Trainee. Present address, Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12181. 7 Career Development Awardee, GM-K3-21,213. An intriguing component of iron-sulfur proteins is a sulfur species known as either inorganic sulfide or acid-labile sulfide. These names originate from observations that various denatura- tive agents liberate inorganic sulfide from these proteins. Spe- cifically, strong acid or base, mercurials, and iron-chelating agents can lab&e the bound sulfur moiety (l-3). To reconstitute these variously denatured proteins a source of inorganic sulfide must be present (3). Alternatively, selenide may be used in place of sulfide to generate an active selenium analogue of the native protein (4). This paper presents data concerning the reaction of the labile sulfide of spinach ferredoxin and other iron-sulfur proteins with the oxidants oxygen and potassium ferricyanide, in which labile sulfide is converted to an oxidized species, apparently covalently bound to the protein and in the formal oxidation state of zero. We call this species sulfur-zero. EXPERIMENTAL PROCEDURE Muter&&--Dithiothreitol, Grade A, was obtained from Cal- biochem; NADH, Grade III, and mersalyll were from Sigma; phenylmercuric acetate, organic analytical standard, was from the British Drug Houses, Ltd. (Toronto) ; sodium hydrosulfite was from Mannox (Hardman and Holden, Ltd.); disodium- 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) was from G. Frederick Smith Chemical Company, Columbus, Ohio; and fresh spinach was from the University of Michigan Food Serv- ice. Cytochrome c was prepared as described by Margoliash and Walasek (5). Ferredoxin NADP reductase and bis-(%- amino-2-carboxyethyl)trisulfide (cysteine trisulfide) were gifts of G. Foust and Dr. J. C. Fletcher, respectively. All other re- agents were reagent grade. To minimize cyanate contamina- tion, urea was incubated at pH 2 for 30 min and then recrystal- lized. All Tris buffers were prepared by dilution of a stock solution of 0.7 M Tris-chloride, pH 7.3 (25’). Details of the preparation of spinach ferredoxin are given elsewhere (6). Its purity is characterized by the value of the ratio (R) of its ab- sorbance at 420 rnp to that at 275 rnp. The highest reproducible value is 0.48 (6), although values of 0.49 have been obtained. Parsley ferredoxin was prepared by a method similar to that used for the spinach protein and adrenodoxin by an unpublished 1 The abbreviation used is: mersalyl, the sodium salt of sodium o-[(3-hydroxymercuri-2-methoxypropyl)carbamylJphenox~a~eti~ acid. by guest on June 3, 2020 http://www.jbc.org/ Downloaded from

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THE JOURNAL OF Bro~oo~ca~ CHEMISTRY

Vol. 246, No. 3, Issue of February 10, pp. 643-653, 1971

Printed in U.S.A.

The Oxygen Sensitivity of Spinach Ferredoxin

and Other Iron-Sulfur Proteins

THE FORMATION OF PROTEIN-BOUND SULFUR-ZERO*

(Received for publication, June 19, 1970)

DAVID PETERING,~ JAMES A. FEE,§ AND GRAHAM PALMERS

From the Department of Biological Chemistry, Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Michigan 48105

SUMMARY

In the presence of urea and molecular oxygen, spinach ferredoxin is rapidly denatured. The product of this reac- tion contains substantial amounts of sulfur in the oxidation state of zero covalently bound to the protein. The origin of the sulfur-zero was shown to be the labile sulfide of the native protein. The S(0) could be released from the pro- tein as S- with a strong reducing agent such as dithio- threitol or as S(0) with cyanide or sulfite anions. Sedi- mentation analysis showed the protein to be polymeric, existing as a dimer.

Ferricyanide could replace oxygen as a source of oxidizing equivalents. With this oxidant the stoichiometry of the reac- tion was found to be 9 moles per mole of ferredoxin. This has been explained by the oxidation of 5 RSH to S/2 RSSR and 2 S2- to 2 S(0). It is proposed that the S(0) is bound to the protein as a trisutide (RSSSR).

The ferricyanide titer of a number of iron-sulfur proteins has been determined and correlated with the S(0) content of the urea-ferricyanide denatured proteins. The oxidation stoichiometries are consistent with a generalization of the above formulation. Thus 8 to 10 moles of ferricyanide are consumed by the two-iron proteins and approximately 30 by the bacterial ferredoxins. With the two-iron proteins, 60 to 80% of the labile sulfide was found as bound S(0) while with bacterial ferredoxins about 50% of the labile sulfide was detected as bound S(0).

In the case of putidaredoxin it was found that the same amount of S(0) was bound when the protein was air-de- natured in the absence of urea and mercaptoethanol as when the protein was denatured in aerobic urea solution. It is proposed that the oxygen sensitivity of many non-heme iron labile-sulfide proteins finds explanation in the oxidation of their mercaptide-sulfide system.

* This work was supported by National Institutes of Health Research Grant GM 12176.

$ National Science Foundation Predoctoral Fellow. Present address, Department of Chemistry, Northwestern University, Evanston, Illinois 60201.

$ National Institute of Health Postdoctoral Trainee. Present address, Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12181.

7 Career Development Awardee, GM-K3-21,213.

An intriguing component of iron-sulfur proteins is a sulfur species known as either inorganic sulfide or acid-labile sulfide. These names originate from observations that various denatura- tive agents liberate inorganic sulfide from these proteins. Spe- cifically, strong acid or base, mercurials, and iron-chelating agents can lab&e the bound sulfur moiety (l-3). To reconstitute these variously denatured proteins a source of inorganic sulfide must be present (3). Alternatively, selenide may be used in place of sulfide to generate an active selenium analogue of the native protein (4). This paper presents data concerning the reaction of the labile sulfide of spinach ferredoxin and other iron-sulfur proteins with the oxidants oxygen and potassium ferricyanide, in which labile sulfide is converted to an oxidized species, apparently covalently bound to the protein and in the formal oxidation state of zero. We call this species sulfur-zero.

EXPERIMENTAL PROCEDURE

Muter&&--Dithiothreitol, Grade A, was obtained from Cal- biochem; NADH, Grade III, and mersalyll were from Sigma; phenylmercuric acetate, organic analytical standard, was from the British Drug Houses, Ltd. (Toronto) ; sodium hydrosulfite was from Mannox (Hardman and Holden, Ltd.); disodium- 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) was from G. Frederick Smith Chemical Company, Columbus, Ohio; and fresh spinach was from the University of Michigan Food Serv- ice. Cytochrome c was prepared as described by Margoliash and Walasek (5). Ferredoxin NADP reductase and bis-(%- amino-2-carboxyethyl)trisulfide (cysteine trisulfide) were gifts of G. Foust and Dr. J. C. Fletcher, respectively. All other re- agents were reagent grade. To minimize cyanate contamina- tion, urea was incubated at pH 2 for 30 min and then recrystal- lized. All Tris buffers were prepared by dilution of a stock solution of 0.7 M Tris-chloride, pH 7.3 (25’). Details of the preparation of spinach ferredoxin are given elsewhere (6). Its purity is characterized by the value of the ratio (R) of its ab- sorbance at 420 rnp to that at 275 rnp. The highest reproducible value is 0.48 (6), although values of 0.49 have been obtained. Parsley ferredoxin was prepared by a method similar to that used for the spinach protein and adrenodoxin by an unpublished

1 The abbreviation used is: mersalyl, the sodium salt of sodium o-[(3-hydroxymercuri-2-methoxypropyl)carbamylJphenox~a~eti~ acid.

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644 Oxygen Sensitivity of Iron-Sulfur Proteins Vol. 246, No. 3

method, the details of which were kindly provided by Dr. T. Kimura. Putidaredoxin was the gift of Dr. J. C. M. Tsibris and Dr. I. C. Gunsalus. Mr. R. Mathews provided the ferre- doxin from Micrococcus luctylyticus and Dr. S. Mayhew the ferredoxins from Clostridium pasteurianum and Peptostrepto- coccus elsdenii.

Preparation of 8=%FerrecZo~in-Four micromoles of ferredoxin (R = 0.48) were treated with a lo-fold excess of mersalyl. Ten minutes later a loo-fold excess of Naz8% and a 25-fold excess of dithiothreitol were added to the bleached protein solution. After 30 min at room temperature, the sample was adsorbed on a DE-52 column (1 X 5 cm), washed with 0.15 M Tris-Cl, and eluted with the buffer containing 1 M NaCI. The protein was then applied to a P-60 Bio-Gel column (2 x 30 cm) which was developed with 0.15 M Tris-Cl. The colored eluate gave R = 0.47 and had 5.3 x lo5 cpm per pmole of ferredoxin.

Analytical ProceduresSulfhydryl-disulfide titrations with mercurial were performed amperometrically with a dropping mercury electrode as described elsewhere (6). Oxidative titra- tions with ferricyanide were performed under identical condi- tions except that the microburette contained a standardized solution of potassium ferricyanide. The latter titrations were also performed spectrophotometrically at 420 rnp with an an- aerobic cuvette equipped with a microburette. The end point was taken as the intersection of the residual absorbance with the ascending slope produced by the absorbance of the excess ferricyanide.

Labile sulfide analyses were done according to a modified Fogo-Popowsky procedure (7). With urea-oxygen-denatured ferredoxin, 20 to 40 nmoles of protein and a 200-fold excess of dithiothreitol were incubated for 15 min in 0.2 ml of solution. Then 0.1 ml of 3 N NaOH was added rapidly to keep the sulfide in solution while the container was opened to introduce the reagents for sulfide analysis. These measurements were im- proved by carrying out the incubation anaerobically in a closed system. Urea-ferricyanide-denatured ferredoxin and cysteine trisulfide were incubated in this fashion. Specifically, 60 nmoles of ferredoxin in 0.05 ml, 1 pmole of dithiothreitol in 0.8 ml of HzO, and 0.05 ml of 3 N NaOH were placed separately in three compartments of an anaerobic vessel. After anaerobiosis, pro- tein and dithiothreitol were mixed and left at room temperature for 30 min. Then base was tipped in, the cell was opened, and sulfide was analyzed. For trisulfide, 440 nmoles of trisulfide and a 45-min incubation were used.

Various spectral measurements were made with Cary 15, Beck- man DU, and Gilford modified DU spectrophotometers, a Jasco W-5 scanning spectropolarimeter equipped to make circular dichroism measurements, and a Varian V-4500 electron para- magnetic resonance spectrometer, equipped with a circulator (8) and with a cryogenic system permitting operation at tempera- tures in the range 740” K.

Amino acid analyses were carried out with a Spinco model 120-C analyzer on samples which had been hydrolyzed in 6 N

HCl under nitrogen for 20 hours at 110”. Molecular Weight Determination-Sedimentation velocity runs

were done on a Spinco model E analytical ultracentrifuge. Dif- fusion coefficients were determined from the schlieren patterns by assuming a gaussian line shape for the peak and utilizing the associated equation for its standard deviation.

~2 = 201

Native ferredoxin was centrifuged in a synthetic boundary cell and oxidant-denatured ferredoxin in a standard 17-mm cell. The partial specific volume was determined by the method of Hunter (9).

Activity Measurements-As a measure of ferredoxin activity, the rate of reduction of cytochrome c by NADPH, catalyzed by ferredoxin NADP reductase and ferredoxin, was monitored spectrophotometrically by following the increase with time of the absorbance at 550 mp of reduced cytochrome c. Specif- ically, 2.7 ml of 0.01 M Tris-Cl (pH 8.0), 0.2 ml of 3.5 x 10-4 M

cytochrome c, 0.1 ml of 10v8 M NADPH, and 0.02 ml of 10-4 M

ferredoxin NADP reductase were mixed, and the reaction was started by the addition of 0.02 to 0.025 ml of 4 x lo+ M ferre- doxin. The reaction was followed at room temperature (23”).

Urea-Oxygen Denaturation-Solid urea was placed into a ferredoxin solution and stirred until dissolved. A relative vol- ume increase of 1.30 occurred with the addition of urea to a final concentration of 5 M, and 1.56 for 8 M urea. The reaction mix- ture stood at room temperature until the initial color had been replaced by the yellow hue of the denatured material and the urea was then removed by dialysis or gel filtration.

Urea-Ferricyanide Denaturation-Two methods have been used for this process. A simple method is to make 2 ml of the ferredoxin solution anaerobic by rapid purging with high purity helium at room temperature for 15 min, at which time solid urea is added. Ten minutes later solid potassium ferricyanide is dropped into the bubbling solution and the mixture is incubated until the color had become bluish (9:1, ferricyanide to protein) or yellow (excess ferricyanide). A trace of the surfactant 2- octanol is present throughout to minimize frothing. The solu- tion is then passed over a column (1 x 15 cm) of Bio-Gel P-2 which was developed with 0.15 M Tris-chloride. The product is freed of iron by addition of a go-fold excess of Tiron and the protein is separated from the iron chelate on a small column of Bio-Gel P-6.

The second method is more cumbersome but there is greater control over the degree of anaerobicity in the system. The ferredoxin is placed in the main body of a multiple side armed Thunberg tube and solid urea and ferricyanide is placed in the side arms. The cuvette is made anaerobic by several cycles of evacuation and purging with inert gas (6) and then the ferre- doxin is tipped into the urea. When this has completely dis- solved the solution is tipped into the side arm containing the ferricyanide and the reaction mixture is then set aside in the dark at room temperature until the requisite color changes have occurred. The vessel is then opened, the Tiron is added, and the protein solution is passed over a Bio-Gel P-6 column (2 x 30 cm) to yield quantitatively the apoprotein free of iron and urea. Although this method of iron removal was satisfactory for spin- ach ferredoxin, a substantial amount of the red ferric Tiron complex migrated with parsley ferredoxin on the Bio-Gel P-6 column. Prolonged incubation with EDTA does remove the iron from all of the proteins examined. The iron can also be removed from the two-iron proteins by direct precipitation of the protein by trichloracetic acid leaving the iron in solution.

Reconstitution of Apojerredoxin-Urea-oxidant ferredoxin was reconstituted with ferric citrate and dithiothreitol (cf. Table IV). When used, sulfide was added as sodium sulfide. After incuba- tion for 30 min at room temperature with occasional shaking, the reaction mixture was passed over a short Bio-Gel P-2 column

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Issue of February 10, 1971 D. Petering, J. A. Fee, and G. Palmer 645

Tet;upreera- PH’

4” 5.2

4 7.6 4 9.1 4 7.6 4 7.6

46 f 2 7.4 63 f 2 7.4 63 f 2 7.4

TABLE I

Stability of ferredoxin

NaCl

.w

0.03 0.45b

0.03 0.45 0.03 0.45 0.03 0.45b 1.0 0.45 0.18 0.43c 0.18 0.43 0.18 0.46d

Initial

-

-_

-

Absorbance ratio

Final The

0.37 4 days 0.14 17 days 0.40 17 days 0.42 17 days 0.40 17 days 0.45 17 days 0.42 47 min 0.0 4 to 6 min 0.38 27 min

0 At room temperature. b Ferredoxin (3.5 X lo+ M) in 0.15 M Tris-Cl (pH 7.6), 0.1 M

borate (pH Q.l), or acetate buffers. 0 Ferredoxin (5 to 6 X 1W4 M) in 0.15 M Tris-Cl. d Under anaerobic conditions.

(1 x 15 cm) to obtain the reconstituted product. The extent of reconstitution was calculated from the equation

Per cent reconstitution = l/R, - 0.66 x 1oo 1/R, - 0.66

where R, and R, are the absorbance ratios of the starting and reconstituted material, respectively (10).

Reaction of Oxidized Apoferredoxin with Thiophiles---aKS- Labeled oxidized apoferredoxin (0.3 ml) was placed in a short sack constructed of 9/32 Visking tubing. The sack was knotted at one end only and the other was slipped snugly over a short length of glass tubing mounted vertically in the plastic lid of a 15-ml cylindrical glass vial. Five milliliters of 0.1 M ethyl- morpholine buffer, pH 8.0, and thiophile (as noted, Table VI) were placed in the vial, the lid was inserted, and the glass tubing was adjusted so that the sack was immersed in the dialysis fluid. The vial was shaken at 23” and samples were removed from the vial and sack (or both) as required for scintillation counting.

Analyses for Sulfur-Zer+Sulfur at the formal oxidation state of zero was estimated as thiocyanate after reaction with cyanide (11) by the technique of Sijrbo (12). Specifically, a sample of apoprotein containing about 0.2 pmole of sulfur-zero was made up to a volume of 2 ml with water, 0.2 ml of N NHPH, and 0.25 ml of 0.1 N KCN. After incubation at room temperature for 40 min, 0.25 ml of Sorbo’s reagent (12) was added, any protein precipitate was removed by centrifugation, and the ferric thio- cyanate formed was determined spectrophotometrically at 460 rnp. One micromole of both sodium thiocyanate and cysteine trisulfide gave an optical density of 1.35 in this assay. External and internal standards of sodium thiocyanate were run in these analyses. The assay is linear up to an optical density of 3.0 (Gilford spectrophotometer).

Iron was removed from the protein prior to analysis to pre- vent ferricyanide formation during the incubation with cyanide. In the case of adrenodoxin in which iron was not removed, no blue color (Prussian blue) was formed. For estimation of pro- tein-bound sulfur-zero by reaction with sulfite, samples were prepared for analysis as follows. One milliiiter of oxidized ferredoxin plus 2 pmoles of sodium sulfite were incubated at

I I I I

TIME (MINUTES)

FIG. 1. Effect of sodium chloride and pH on the stability of spinach ferredoxin in urea solution. Left, effect of sodium chlo- ride. Ferredoxin (9.8 X 10-S M) was incubated at room tempera- ture in 5 M urea-O.15 M Tris-Cl, and various concentrations of sodium chloride as shown and the absorbance at 420 rnp was re- corded at intervals. The relative oxygen concentrations are 1.0, 0.89, and 0.83 for 0.5, 0.7, and 1.04 M NaCl, respectively (Znterna- tional Critical Tables, Vol. ZZZ, p. 272). Right, ferredoxin (1.06 X 1W4 M) was incubated at room temperature in 8 M urea, 0.19 M

NaCl, and 0.01 M Tris-chloride at the pH values shown and the absorbance at 420 rnp was recorded at intervals.

room temperature for 4 hours. The thiosulfate was separated from the protein either by precipitation of the protein with 0.05 ml of 20% perchloric acid or by dialysis of the sample against a known volume of 0.1 M N-ethyl-morpholine buffer, pH 8.0, which was then lyophilized and the residue was dissolved in 2 ml of water. The amount of thiosulfate was then determined as thiocyanate as described by Sorb0 (12). In this procedure 0.5 pmole of sodium thiosulfate gave an absorbance of 0.565 at 460 rnp.

RESULTS

Stability of Ferrer&x&-Table I contains a brief summary of the effects of pH, sodium chloride, concentration, and tempera- ture upon the stability of ferredoxin, defined here as the resist- ance of the protein to bleaching and measured as a decrease in the absorbance ratio R. The deterioration of ferredoxin at acid pH and in solutions of low sodium chloride concentration is well known (lo), and these conditions should be specifically avoided in storage. Although the spinach protein may be kept indefi- nitely in 1 M sodium chloride, pH 7.9, at O”, alfalfa ferredoxin cannot be stored aerobically without loss of color. However, this protein is stable for extended periods anaerobically (13). At elevated temperatures oxygen sensitivity is also noted with spinach ferredoxin. In particular, the comparison of bleaching at 63” in the presence and absence of oxygen indicates that the primary effect of temperature is to accelerate the reaction of oxygen with the protein. The data at 46” may be compared with the 26% decrease in absorbance ratio for alfalfa ferredoxin in 0.1 M sodium chloride and 0.01 M Tris-chloride, pH 7.5, at 48” to point out again the relatively greater sensitivity of the alfalfa protein to oxygen denaturation (13).

Properties of Ferreckin in 6 M Urea-When ferredoxin is dis- solved anaerobically in 5 M urea, the absorption at 420 rnp de- creases less than 5% over a period of 24 hours. In high sodium chloride the optical and circular dichroism spectra resemble those

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646 Oxygen Sensitivity of Iron-Suljur Proteins Vol. 246, No. 3

TABLE II

Properties of urea-ox3

Molar extinction coefficient at 275 m/.t

Iron protein” (Iron free)b

Circular dichroism spectrum Electron paramagnetic reso-

nance spectrum Mercurial titer

en-denatured ferredoxin

20,000 f 1,000 liters per mole-cm 13,000 f 1,000 liters per mole-cm None, 320 to 700 rnr None, oxidized, or reduced

2.4 f 0.1 in presence of sulfite; 4 samples; R = 0.45 to 0.46; 0.0 in absence of sulfite

a AZ15 determined after urea denaturation of a known con- centration of ferredoxin.

b An5 determined after either quantitative dialysis or Bio-Gel separation of iron-Tiron chelate from a known amount of urea- oxygen-denatured ferredoxin (R = 0.46 to 0.47).

of the native protein while in low sodium chloride there are sig- nificant reversible changes in both the optical and circular di- chroism spectra (6). On addition of oxygen (air) to the solu- tion, rapid bleaching of the visible color occurs. The kinetics of this bleaching has been measured as a function of sodium chloride concentration and pH and some of the data recorded in Fig. 1. There is a marked decrease in the rate of bleaching as the sodium chloride concentration increases but this effect is complicated by the fact that the concentration of dissolved oxygen varies with salt concentration. However, the effect of the sodium chloride far exceeds that accounted for by the de- crease in oxygen concentration in this series. This assumes that the presence of urea does not magnify these oxygen differ- ences. The pH dependence of bleaching shows a definite sta- bilization of the protein against oxidation as the pH is increased. These curves resemble those for the urea-oxygen or guanidine hydrochloride-oxygen bleaching of adrenodoxin (14).

Properties of Urea-Oxygen-denatured Product-Upon comple- tion of bleaching, the product was characterized in several ways, as summarized in Tables II and III. The material has a pale yellow color, and only exhibits an end absorption in the visible region of the spectrum. The extinction coefficient of the 275 mp peak in the native protein is not changed by denaturation. It does diminish significantly after iron is removed (Table II). The iron shows no electron paramagnetic resonance signal either in the presence or absence of the reducing agent, sodium hydro- sulfite. Furthermore, the iron is not free in solution as judged both by a slow appearance of the iron-Tiron color and by the inability to remove iron from the denatured protein by dialysis or gel filtration.

From the mercurial titer of the denatured protein, it is clear that no sulfhydryl groups remain free, for only in the presence of sulfite does the titrant become bound. The disulfide titer of 2.4 (Table II) implies that the protein has polymerized so that at least one of its five sulfhydryl groups participates in an inter- molecular disulfide bond.

A comparison of the molecular weight of both native ferredoxin and the urea-O2 denatured ferredoxin is presented in Table III. There is good agreement between the results on the native pro- tein and the molecular weight of 10,660 calculated from the amino acid composition (28). Both the sedimentation and diffusion coefficients are similar to those obtained for alfalfa ferredoxin (13). The partial specific volume is significantly

TABLE III

Molecular weight of ferredoxin

Spinach ferredoxin

Native Urea-02 Alfalfa

(denatured) Urea-Fe(CN)&

(denatured) ferredoxirP

szo,lu x 10’3 1.68 f 0.05b 2.0 f O.lc 2.W 1.58 (se@)

D 20,w 107 (cm2 see-I)

11.9 f 0.7b 9.4 f 0.6~ 9.w 10.4

B(ml mg-i) 0. 691e 0.71 0.71 0.715 (assumed) (assumed)

Molecular 11,000 18,000 19,000 12,900 weight f 1,000 f 2,000

0 These results are taken from Reference 13. b Five SZQ+ and three Dzo,~ determinations on protein with

ratios of 0.43 or 0.46, ranging from 3.33 to 6.64 mg per ml, com- prise these averages. The medium was either 0.05 M KCl, pH 7.3, or 0.1 M KCl, 0.01 M Tris-Cl, pH 7.4. Runs were done at 20’ at a rotor speed of 59,760 rpm. The error in each value is the range of the data. No concentration dependence of the coefficients was noted.

c Average of six runs, protein has initial ratio of 0.46 to 0.47. d Average of two runs at 8 and 10 mg per ml. o An average of two values: 0.695 for 6.65 mg per ml of fer-

redoxin at 23.24’ and 0.688 for 5.78 mg per ml at 22.33”.

lower than had previously been realized. It was determined by the very precise method of Hunter (9). During sedimentation- velocity experiments the denatured protein exhibited a single symmetric schlieren pattern and the analytical data (Table III) are consistent with its existence as a dimer, a value of 18,000 being determined for the molecular weight. The urea-oxygen product elutes as a single symmetrical peak from a Sephadex G-75 column while disc gel electrophoresis reveals the presence of multiple components. Hence, although the product appears to be homogeneous with respect to size, there is extensive poly- dispersity presumably due to the presence of species with differ- ent charge characteristics.

The uniqueness of the urea-oxygen denaturation is revealed in the ability to reconstitute the native protein from this oxidized material in the absence of an external source of sulfide. In Table IV typical results of reconstitution experiments with the stand- ard reagents are collected. It is clear from the data that sub- stantial reconstitution can be obtained with iron and dithio- threitol alone, about 55 ‘% reconstitution being realized with these reagents. Further addition of sodium sulfide improves the yield to about 80%. Use of 8 M urea, higher oxygen concentra- tions, lower temperature, or various concentration of reagents in the reconstitutions did not improve this value. We have not explicitly studied the effect of varying the incubation time during reconstitution. In the examples in Table IV, the protein was either dialyzed, passed over a Bio-Gel P-60 column, or even ex- tracted with carbon disulfide to minimize the possibility that unbound sulfur in some form remained in the reconstitution mix- ture.

The oxidative denaturation apparently leads to significant binding of labile sulfide to the protein although the bound sulfur species is not detected by the standard labile sulfide assay. However, after prior incubation of the apoprotein with dithio- threitol, an amount of labile sulfide is found in quantitative

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Issue of February 10, 1971 D. Petering, J. A. Fee, and G. Palmer

TABLE IV Reconstitution of urea-oxygen denatured ferredoxin

647

DenaturatimP

[Fdl R [N&l] IF4 Dithio- threitol

x 10’ dl M x 10’ M

4.5 0.41 0.77 4.50 25

1.6 0.48 0.2 4.7d 25 25

6.5 0.47 0.77 4.OJ 25

-

.-

-

Fe R reconstituted

s- Fd

1 0.20

2.5 1 I 1 I 0.27

-

% % 60

46 4* 81

60

@ Other conditions: 0.15 Y Tris, pH 7.5, 5 M urea. b Other conditions: 0.15 M Tris, pH 7.5, room temperature, ferredoxin was approximately 10-d M. c Ferredoxin in the 5 M urea denaturing solution, iron not removed. d This sample was freed of iron by overnight incubation with Tiron followed by Bio-Gel P-10 chromatography. The product was

iron-free by analysis. 6 Determined as labile sulfide after treatment with dithiothreitol (cf. “Experimental Procedure”). f Protein is urea-free but contains iron. It was extracted three times with v/v portions of CS2.

agreement with the corresponding degree of reconstitution of the sample (Table IV). It seems, therefore, that the restricting fac- tor in reconstitution resides in the supply of bound sulfur. Hence, when external sulfide is provided, the extent of the recon- stitution is significantly increased (Table IV). The reconstitu- tion in the presence of urea confirms the observations (6) that urea, itself, does not destroy the chromophore site and that the effect of oxygen is reversed in the presence of dithiothreitol.

The isolated product, taken from the first reconstitution ex- periment of Table IV and purified by ammonium sulfate pre- cipitation was subjected to a number of tests for the identification of ferredoxin (Table V, Figs. 2 and 3). By assuming that recon- stituted and native proteins have the same extinction coefficient at 420 rnp, several properties of these two species have been com- pared directly. It is evident that these two materials have iden- tical chromophores with both the optical and circular dichroism spectra of the oxidized and the electron paramagnetic resonance spectrum of the reduced protein being identical with the native species. The iron and labile sulfide analyses give the expected stoichiometry, and the reconstituted protein is fully active in the coupled TPNH reductase-cytochrome c assay (Table V) .

Denaturattin with Ferricyanide in Presence of Urea-The oxi- dative denaturation of ferredoxin has been examined with a sec- ond oxidant, ferricyanide. Although native ferredoxin does not react with ferricyanide in a 5-fold excess, in anaerobic urea it does consume oxidative equivalents. When followed amperometri- tally (Fig. 4), the reaction has a well defined end point of 9 moles of ferrioyanide per mole of protein. The slope of the ascending portion of the titration is the same as in the absence of protein; i.e. further reduction of ferricyanide is not taking place. A spec- trophotometric titration gives a titer of 9.1 moles and, although the kinetics of bleaching of the visible spectrum by this oxidant was complex with both fast and slow phases, the end point is well defined. The number of moles of ferricyanide reduced by several iron-sulfur proteins has been determined and the data are col- lected in Table VI. Clearly the experimental titers are in agree- ment with the predicted values assuming that each mercaptide consumes 1 oxidizing eq, each sulfide consumes 2, and in the bacterial ferredoxin, that 4 of the 8 iron atoms react as ferrous ion (15).

TABLE V

Properties of reconstituted ferredoxin*

Optical spectrum Identical with native of the same R (Fig. 2)

Circular dichroism spectrum Identical with native between (Fig. 3) 320 and 700 rnp

Electron paramagnetic reso- Identical with native, having nance spectrum apparent gvalues; g, = 1.896,

g, = 1.956, g. = 2.046 Iron content per mole of recon- 2.06 atom@

stituted product Labile sulfide content per mole 1.89 atom@

of reconstituted ferredoxin Activity per Amb Identical with native fer-

redoxin Native 635 pmoles of cytochrome c

reduced per min Reconstituted 695 pmoles of cytochrome c

reduced per min Urea-02 Fd 8.25 amoles of cytochrome c

reduced per min

a Denaturation in 5 M urea; reconstitution from the same solu- tion with 25:l dithiothreitol-Fd (R = 0.30); purification by am- monium sulfate precipitation to R = 0.38.

b The molar extinction coefficient of reconstituted ferredoxin was assumed to be 9400 liters per mole-cm.

As with the urea-oxygen-denatured ferredoxin, a nonintegral mercurial-suhite titer was obtained with the urea-ferricyanide product. After treatment with 9 moles of ferricyanide, 2.6 moles of disulfide were found by mercurial-sulfite titration. The iron- free product of ferricyanide denaturation was titrated similarly and 2.7 moles of disulflde found (assuming a ~276 = 13,000 liters per mole-cm for the apoprotein (Table II)) suggesting the pres- ence of polymeric material cross-linked with disulfide bonds. Similarly, the molecular weight as measured in the ultracentri- fuge supports the postulate of a dimerized product (Table III). However, the material was polydisperse on Sephadex G-75 elut- ing with a minimum of three peaks and multiple bands were also observed on disc gel electrophoresis.

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FIG. 2. Comparison of the optical spectra of native (X) and reconstituted (-) spinach ferredoxin. The data for the native protein are for an aged sample which had a value for R similar to that of the reconstituted samole: conditions. 0.15 M Tris-chloride-l

* I

M NaCl at 23’.

1 I I

400 500 600 WAVELENGTH - mp

FIG. 3. Comparison of the circular dichroism spectrum of native (X) and reconstituted (--) spinach ferredoxin (cf. Fig. 2).

The urea-ferricyanide-denatured product can also be recon- stituted without added sulfide (Table VII) with an average yield of approximately 85 y0 which is significantly higher than that ob- tained with the oxygen product (55%). Dithiothreitol releases all of the bound sulfur (Fig. 5, Table VIII) and the amount of sulfide liberated is the theoretical 2 moles per mole of protein (Table VIII).

The ability to be reconstituted seems to be insensitive to the conditions of denaturation, for variations in the concentrations of urea, sodium chloride, and ferricyanide in the denaturation medium have little effect on the yield of reconstituted protein (Table VII).

As described under “Experimental Procedure” one can recon- stitute mercurial-treated ferredoxin with Na$%. The ferredoxin so obtained is highly radioactive with the radioactivity confined exclusively to the labile sulfide moieties of the protein with a spe- cific activity of 5.3 x lo6 cpm per pmole of protein. When this material is subjected to the urea-ferricyanide denaturation pro- cedure, the pooled fractions from the Bio-Gel P-6 column had 4.8 X lo6 cpm per fimole of protein assuming quantitative re-

I I I I / I 2 4 6 8 IO 12

MOLES FERRICYANIDE ADDED PER MOLE SPINACH FERREDOXIN

FIG. 4. Amperometric titration of spinach ferredoxin with potassium ferricyanide in 5 M urea under anaerobic conditions (cf. “Experimental Procedure”).

covery of protein; i.e. 90% of the radioactive sulfide was retained in the oxidized apoprotein.

The formal description of the oxidation-reduction-active com- ponents of the native protein is two Fe(III), five sulfhydryl, and two sulfide groups as determined from mercurial titrations fol- lowed by iron valence determinations (1). The conversion of 2 moles of sulfhydryl to 1 mole of disulfide requires 2 eq of oxidant while iron is not expected to be further oxidized. Therefore, given the stoichiometry of the ferricyanide titration (9 : l), the disulfide content (2.5), and the nearly quantitative production of bound sulfur, 4 eq of ferricyanide may be assigned to the oxida- tion of the sulfide ions, raising them to the formal oxidation state of zero (sulfur-zero). The fact that the reducing agent, dithio- threitol, liberates bound sulfur as sulfide supports the proposal of an oxidative binding of labile sulfide (Tables IV and VIII).

The possible modes of binding of sulfur-zero in proteins are re- stricted by the limited variety of amino acid functional groups. However, there is one conspicuous possibility: its binding by sulfhydryl residues (II), as a trisulfide. Three important obser- vations support this possibility. First, cysteine trisulfide yields S2- in 77% yield when incubated with dithiothreitol. Second, mercurial analysis of cysteine trisulfide in the presence of sulfite is 0.8 mole per mole, showing that disulfide and trisuhide behave similarly in this reaction (Table VIII). Third, cysteine trisulfide is not subject to further oxidation by small excesses of ferricya- nide under the conditions of our experiments as determined am- perometrically. Hence, it is possible to oxidize the labile S*- to S(O), and, if the S(0) is bound as a trisulfide, to have it resistant to further oxidation by ferricyanide.

Mobilization of Sulfur-Zero by ThiophilesThe possible reac- tions in the breakdown of a trisulfide (RSSSR) by a suitable nucleophile (X-) are (16)

RSSSR + X- + RSSX + RS- (1)

RSSX + X- --) RSX + SX- (2)

RSSX + RS- + RSSR + SX- (3)

One of the products will be of low molecular weight (SX-) and thus rapidly diffusible across a membrane. We have therefore

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TABLE VI

649

Stoichiometry of ferricyanide oxidation and sulfur-zero content of iron-sulfur proteins

Protein

Spinach ferredoxin Spinach ferredoxin Spinach ferredoxin Spinach ferredoxin Spinach ferredoxin Spinach ferredoxin Spinach ferredoxin (trichlorace-

tic acid apoprotein) Parsley ferredoxin Parsley ferredoxin Putidaredoxin Putidaredoxin

Adrenodoxin

Bacterial ferredoxins Micrococcus lactylyticus Clostridium pasteurianum Peptostreptococcus elsdenii Peptostreptococcus elsdenii

.- Foundb

Ferricyanide titer

CalculatedG Oxidantd

moles/mole @&2in

8.0

9.6

10.1

30.7 28 25

9h

8

9

280) 28 28’

Fe (CN) ,$- Fe (CN) &a- Fe (CN) e* Oxygen Oxygen Oxygen

1.25, 1.61f 2 -g

1.26, 1.71 2 --I

1.66 2 4

0.96 2 --P

1.1 2 --o

1.52 2 4

0.0 0 Iron Free

Fe (CN) s8- Fe (CN) g8- Oxygen Oxygen (no

urea) Oxygen

1.32 2 -5

1.36 2 -c

1.42 2 -i 1.46 2 --i

1.2 2 Iron not re- moved

Oxygen 3.36 8 -.A

Fe (CN) 6a- 3.85 8 2

Fe (CN) gs- 4.6 8 A

Fe (CN) 6a- 5.5 8 --k

Method of iron removal

(1, 10, 27)

(27)

(4)

(28)

(15, 22, 29) (30)

a Determined with cyanide as thiophile except as shown (cf. “Experimental Procedure”). 5 Spinach ferredoxin: 40~1 of SFd solutionwith A 4~0 = 10.73, ~420 = 9,400 liters per mole-cm, A~z~:A~x = 0.47. Parsley ferredoxin:

15 ~1 of PFd solution with AIZZ = 26.85, ~1.~2 = 9,200liters per mole-cm, Atzz:Az,, = 0.625. Putidaredoxin: 250 ~1 of putidaredoxin solu- tion with Atso = 1.61, ~150 = 9,400 liters per mole-cm. Adrenodoxin: 70 ~1 of adrenodoxin solution with A165 = 4.96, ~46~ = 9,800 liters per mole-cm, A~~~:ALwI = 0.693. Micrococcus lactylyticus: 100 ~1 of ferredoxin solution with Aas0 = 2.25, ~3~0 = 30,000 liters per mole-cm, Aw:Az~~ = 0.77. Clostridium pasteurianium: 10 ~1 of ferredoxin solution with A 390 = 28.3, C880 = 30,000 liters per mole-cm, Aa,o:Azso = 0.793. Peptostreptococcus elsdenii: 5 ~1 of ferredoxin solution with A 380 = 56.8, ~390 = 30,000 liters per mole-cm, Aaeo:Azao = 0.80. Titrations were carried out with 6.45 mM K8Fe(CN)6 solution as described in Reference 6.

c Calculated from the cysteine and sulfide content assuming a stoichiometry of one ferricyanide per cysteine, two ferricyanides per sulfide moiety and in the bacterial ferredoxins, one ferricyanide per each of the 4 ferrous ions.

d The oxidations with ferricyanide were carried out with a 10% expected equivalent excess in 4 to 6 M urea solutions under anaerobic conditions. Generally 15 to 30 min are required for the reaction to go to completion at room temperature. Oxidations with molecular oxygen were carried out in air-saturated solutions of 4 to 6 M urea. These reactions were allowed to proceed for 10 to 24 hours.

6 Equated with labile sulfide content. f Determined with sulfite as thiophile (cf. “Experimental Procedure”). o Tiron was added to the final reaction mixture in excess of iron. After approximately 1 hour the solution was passed over a Bio-

Gel P-6 column, and the protein-containing fractions were pooled and freeze-dried. h Parsley ferredoxin contains five half-cysteines per molecule (S. Keresztes-Nagy, personal communication). i The final reaction mixture was brought to 8% trichloracetic acid. The precipitated protein was centrifuged down and redissolved

in Tris buffer containing lo-2 M EDTA. This process was repeated three times with the EDTA eliminated in the final dissolution of the protein.

j Same as Footnote F, except that no EDTA was used. k The final reaction mixture was brought to W2 M EDTA and allowed to stand until the blue or blue-green color dissipated. The

solutionwas passed over a Bio-Gel P-2 column. The protein-containing fractions were pooled and freeze-dried. 1 P. elsdenii ferredoxin contains 7 to 8 half-cysteine per molecule (S. G. Mayhew, personal communication).

attempted to release radioactive sulfur from the protein with and the dialysis fluid. With dithiothreitol, on the other hand, nucleophiles and to identify the product. Nucleophiles such as the radioactivity rose initially but after 4 hours fell again to a cyanide, sulfite, and mercaptides which react well with sulfur in steady low value (Fig. 5). Cysteine was intermediate in that

the zero valent state are generally termed thiophiles (17). about 55% of the radioactivity was found in the dialysis fluid. When the Y+urea-ferricyanide-oxidized protein was incubated After 50 hours of incubation when both sack contents and dialy-

with various thiophiles, the radioactivity was rapidly released sis buffers of the various samples were analyzed, all except the (Fig. 5). The yield of radioactivity that appeared in the dialysis control showed virtually complete equilibration of the radioac- fluid varied considerably with the different nucleophiles. Thus, tivity. The control sample retained almost all of its radioac- both sulfite and cyanide gave quantitative recovery of radioac- tivity. The slow appearance of radioactivity in the dialysis

tivity assuming complete equilibration between the sack contents medium (Fig. 5) is attributed to the low but finite porosity of the

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TABLE VII

Reconstitution of urea-ferricvanide-denatured jerredoxin

Denaturation: [Fd], solvent

2.3 x lo-’ M Fd R = 0.47

0.4 M N&Cl 8 M urea

1.1 X lO+ M Fd R = 0.46 0.8 M NaCl 5 iv urea

- Denatura-

tion’“: ?e(CN)&:

Fd

175: 1

7O:l

9:l

Reconstitutio& [Fdl X ‘Of, M

dith$~thr~tol:

0.85 60:3:1

1.4 25:4: 1

2.1 25:2.5:1

R reconstituted

0.41

0.42

0.41

ieconstitu- tion

%

84

a7

85

a Buffer is uniformly 0.15 M Tris-Cl, pH 7.5. * Protein is iron- and urea-free unless noted, in 0.15 M Tris-Cl,

pH 7.5; room temperature. All reconstituted samples (1 to 2 ml) were passed over Bio-Gel P-2 column.

dialysis membrane to small proteins. Under the conditions of the experiment (cj. “Experimental Procedure”), it is likely that the rate-limiting step is the diffusion of the mobile species across the membrane, and thus differences in the relative rates of ap- pearance of radioactivity in the dialysis fluid with the different nucleophiles may not be significant. If the mechanism of the release of radioactivity is as predicted in Equations 1 and 3, one would anticipate that mercaptide-trapping agents could inhibit the release of radioactivity from the oxidized protein. Thus p-chloromercuribenzene sulfonate strongly inhibits the release of radioactivity when cyanide is used as the thiophile though iodo- acetamide and N-ethyl maleimide have no significant effect. This effect of p-chloromercuribenzene sulfonate, however, is an indirect one and is due to the formation of a strong complex (Kdiss <4 X 10v6 M) between p-chloromercuribenzene sulfonate and cyanide, thus lowering the amount of cyanide available for reaction. This complex can readily be detected as an absorp- tion maximum at 275 rnp in difference spectra between p-chloro- mercuribenzene sulfonate and p-chloromercuribenzene sulfonate plus cyanide. A weak complex has also been detected between p-chloromercuribenzene sulfonate and sulfite (K&s = 0.04 M, x - 274 mp). max - Sulfite reacts quite rapidly with iodoacetamide Q = 0.035 M+ see-l at 25”, pH 8.0) with presumed elimination of iodide by the sulfonyl group. The absence of any inhibition by either iodoacetamide or N-ethyl maleimide suggests that the re- lease of radioactivity proceeds principally through Equations 1 and 2 of the above reaction scheme.

Quantitative Estimation of Xulfur-Zero-The anticipated prod- uct from the reaction of cyanide (X- = CN-) with oxidized ferredoxin is thiocyanate. Fletcher and Robson (11) have de- scribed a method for assaying the central sulfur of cysteine tri- sulfide as thiocyanate after release by cyanide. When oxidized spinach ferredoxin is assayed by this method we find 0.96 to 1.66 moles of SCN per mole of protein (Table VI). Authentic cys- teine trisulfide gives 1 .O mole of SCN per mole with sodium thio- cyanate as a standard (Table VIII). The higher values are ob- tained with ferricyanide-oxidized protein although there are con- siderable variations in preparation to preparation. An indirect

/ I 70

/ I / I

o . Infinite l

Equilibmtion

IO

r P.- x

1-p ,I n

I I P I IO 20 30 40 50 60

TIME (HOURS1

FIQ. 5. Time course of the release of radioactivity from 36S- oxidized spinach ferredoxin by (O---O) cyanide, (O---O) sulfite, (O----O) cysteine, (X--X) dithiothreitol, (A--A) control. These reagents were present at 5 mM. Samples were removed from the vials at the times indicated and added to a scintillation fluid prepared as described by Gibson, Palmer, and Wharton (16). The samples were counted for 40 min at room temperature; the background was 1280 counts per 40 min. The abscissa is expressed as counts above background per ml per 40 min. At the end of the experiments the radioactivity in each sack was determined; in each case it was a little higher than that anticipated for complete exchange, but in no case was the excess radioactivity greater than 8yo of the initial radioactivity present compared with the 5% predicted for complete exchange. Infinite equilibration represents the counts anticipated when the level of radioactivity is the same in dialysis sack and fluid. For other details see “Experimental Procedure.”

TABLE VIII

Comparison of the reactivity of urea-ferricyanide-denatured jerredoxin with trim&de of cysteine

Removal by X’= Mercurial titer in

XzCN-1 X=S&= 1 “;+;;“- presence of suh5te

% Urea-ferricyanide

ferredoxin . . 100” 100” 1OOb 2.67 Urea-ferricyanide

ferredoxin . 60 to 80~ 80 to 85” lOoa Trisulfide. . 100’ 1OOe 77‘ 0.78”

a Assuming 2 moles of bound sulfur per mole of ferredoxin and 1 mole of bound sulfur per mole of trisulfide.

* See Fig. 5. c See Table VI. d Labile sulfide analysis (cf. ‘LExperimental Procedure”). 0 The trisulfide was analyzed directly as described under “Ex-

perimental Procedure.”

determination has also been made for thiosulfate formation when sulfite is used as the nucleophile (Table VI) ; 1.6 to 1.7 moles of thiosulfate were found even though the particular samples of protein gave only 1.25 moles of thiocyanate.

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Several other iron proteins have been analyzed for their sulfur- zero content and in all cases substantial yields of thiocyanate were obtained. Of particular importance is the observation that putidaredoxin, denatured by exposure to air in the absence of the preservative2 mercaptoethanol, yielded the same amount of thiocyanate as the protein denatured by urea-oxygen in the pres- ence of mercaptoethanol. In general the yields obtained with the bacterial ferredoxins were close to 50%; the possible signifi- cance of this observation is discussed below.

Attempted Direct Demonstration of Cysteine Trisuljide in Oxi- dized Protein-We have tried to demonstrate directly the pres- ence of cysteine trisulfide in hydrolysates of the oxidized protein with both acid and enzymic hydrolysis. Unfortunately this ap- proach is complicated because cysteine trisulfide is unstable under acid hydrolysis (11)) and it migrates on the amino acid analyzer with the same mobility as leucine under standard conditions.3 To overcome this problem, in part, we used oxidized protein in which the sulfur-zero was radioactive, manually collected the ef- fluent from the amino acid analyzer, and examined these frac- tions by scintillation counting. After acid hydrolysis almost all of the radioactivity which is observed in the effluent (--40%) is found in the breakthrough volume; occasionally a trace of radio- activity has been found under the leucine peak. No significant amount of radioactivity was detected in any other fraction.

Normally under the conditions of acid hydrolysis, cysteine tri- sulfide partially breaks down to cysteine though about 50% of the trisulfide remains after 22 hours hydrolysis. However when 0.25 pmole of trisulfide was acid hydrolyzed for 22 hours together with 0.125 pmole of each of the amino acids found in the stand- ard calibrating mixture for the amino acid analyzer (Beckman No. 312220), no trace of trisulfide could be found though there was a significant loss of phenylalanine (50%) and a substantial increase in serine (300%) together with the appearance of trace amounts of unidentified compounds.

Enzymic hydrolyses of the oxidized radioactive protein have been attempted with weight for weight quantities of pepsin, Pro- nase, trypsin, chymotrypsin, and carboxypeptidase A in several combinations. After incubation the mixture was lyophilized and extracted several times with acetone-ethanol-ether

(10: 10: I). No radioactivity was ever detected in the organic extracts. Presumably the trisuliide is part of an undigestible core. We are continuing our attempts to demonstrate the pres- ence of the trisulfide directly.

DISCUSSION

The results presented in this paper provide strong evidence for a novel reaction in the chemistry of iron-sulfur proteins, in which the labile (or inorganic) sulfide moiety is oxidized to the zero valent oxidation state and covalently bound to the protein. This

occurs as the result of an oxidative process which can proceed aerobically and occurs spontaneously to a greater or lesser extent in many of these proteins, but which is best studied by the con- trolled addition of ferricyanide under anaerobic urea conditions. Because of the methods of preparation of thii derivative, we find it advisable to refer to it as either the urea-oxygen- or urea- ferricyanide-denatured protein. However, we stress that the

role of the urea is to facilitate a reaction which has a tendency to proceed even in the absence of this reagent, and we will later

* J. Tsibris and I. C. Gunsalus, personal communication. 3 J. C. Fletcher, personal communication.

document the known intrinsic oxygen sensitivity of many of the members of this class of proteins.

The conclusion that the sulfide can be oxidized and bound to the protein is based on several pieces of evidence. When a%- sulfide-labeled spinach ferredoxin is oxidized with ferricyanide in the presence of urea and the protein is isolated by Bio-Gel chromatography, about 90% of the radioactivity remains asso- ciated with the protein. The oxidized iron-free apoprotein as isolated does not give any reaction in the assay for sulfide unless previously treated with dithiothreitol, when approximate stoichi- ometric values for sulfide are obtained. This apoprotein can be reconstituted to the native protein by the addition of Fe3+ and dithiothreitol alone; an added source of sulfide is not neces- sary. The yield of reconstituted protein appears to be propor- tional to the S2- content of the apoprotein determined after previous treatment with dithiothreitol. This conclusion is strengthened by the fact that procedures which ordinarily sep- arate large and small molecules, such as gel filtration and di- alysis, have no effect on the above results.

Of equal importance is the fact that extraction of the protein solution with carbon disulfide, a good solvent for elemental sul- fur, does not prevent the reconstitution of the protein, precluding the possibility that labile sulfide has been oxidized to unbound elemental sulfur, which, perhaps because of its polymeric struc- ture, is not separated out by the techniques mentioned above.

The conclusion that in this bound form the sulfur is at the formal oxidation state of zero also stems from several experi- ments. The stoichiometry of oxidation of spinach ferredoxin by ferricyanide is 9 moles per mole of protein. This can be explained by the sum of the following reactions assuming the formal description of the oxidized protein is as described above:

5 KS- + 5 Fe (CN) c3- + 5/2(RSSR) + 5 Fe (CN) & (4)

2S- + 4 Fe (CN) 63- + 2 “S”” + 4Fe (CN) & (5)

The reaction of the bound sulfur with the sulfur-reactive nucleophiles (thiophiles) cyanide, sulfite, and mercaptide is strongly suggestive that the sulfur is bound and points more directly to the binding as sulfur-zero. Each of these reagents rapidly and quantitatively liberates bound (radioactive) sulfur from the oxidized protein under very mild conditions. For instance, 5 mu cyanide releases all of the radioactive sulfur in 4 hours at 25” and pH 8.0. By contrast it requires 50 mM cyanide in 0.03 N NaOH at 37” for 30 mm for complete release of the sulfur from the disulfide bonds of insulin (18).

The general reaction of sulfur in the oxidation state zero with thiophiles (X-) is (17)

RS,- + nXm- + RS- + nX-Sm- (6)

in which X-S will be thiocyanate or thiosulfate, respectively, when X is cyanide or sulfite. Hence the demonstration (Table VI) that both thiocyanate and thiosulfate are liberated from the oxidized protein in yields approximating the sulfide content of the protein is strong evidence for the proposal that the sulfur is bound as S(0) in this denatured state.

A plausible extension of this assignment is our proposal that the sulfur-zero moiety is actually bound as a polysulfide (i.e. a tri- or tetrasulfide) group, the outer sulfurs of which are donated by cysteinyl groups on the protein. This is reasonable both because polysuhide groups are a common source of sulfur in this oxidation state (17) and, because, with the limited number

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of protein functional groups to choose from, the polysulfide group is the most likely candidate.

The results of Table VIII are consistent with this identifica- tion. The genuine &sulfide of cysteine behaves as predicted in the cyanide and sulfite-mercurial reactions. The general reaction for polysulfides is then

RS (S),SR + X- -+ RS (S),X + RS-

RS(S),X + nX- -j RSX + nSX-

in which n = 1 for the trisulfide.

(7)

@I

In the presence of an excess of reactive X-, the formation of RSSR (Equation 3) will be suppressed and a single thiol will be released from each polysulfide molecule (17). Therefore, in mercurial-amperometric titrations of polysulfides with different values of n, all should have the same stoichiometry of 1 mole of mercurial bound per mole of polysulfide. This is the case for disulfides (n = 0) and for the model trisulfide (n = 1, Table VIII). Hence the observed values of 2.4 moles of disulfide per mole of protein (Table IV) should in fact be reported as 2.4 moles of disulfide plus polysulfide per mole of protein.

The release of sulfide by dithiothreitol from both the trisulfide (Table VIII) and the oxidized protein (Table IV, Fig. 5) may be explained by the reaction sequence below.

RSSSR + SLS- -+ RSS-S>- + RS-

RSS-SJ + S;S- --f RS-Sj- + -SSJ- (9)

-s-s,- + sz- + s>

Because our efforts to provide direct evidence for the presence of cysteine trisulfide in this protein were unsuccessful there is an ambiguity as to whether the sulfur-zero is bound as either the tri- or tetrasulfide. The consistently low yields of sulfur-zero found with the bacterial ferredoxins are circumstantial evidence that the trisulfide is the principal product. These proteins only contain 8 cysteine residues; if tetrasulfide was the principal product then one would anticipate higher yields of sulfur-zero. Conversely, if the trisulfide alone is formed, the yield could not exceed 50%. With one exception we find yields close to 50%, implying that the trisulfide is the dominant product. However, it could be argued that the efficiency of the oxidation process is lower in the bacterial ferredoxins and that the yield of the re- action is unrelated to the nature of the products. This is in- consistent with the ferricyanide titers for these proteins which agree rather well with the theoretical values predicted on the basis that all of the sulfide is oxidized to sulfur-zero (Table VI). Presumably the oxidized sulfur which is not incorporated into the protein is released into the medium as elemental sulfur. These points remain to be settled.

The reconstitution of ferredoxin from iron-free apoproteins has been achieved in several ways. Thus, mercurial-treated protein which is free of both iron and sulfide can be reconstituted on addition of iron, sulfide, and a thiol which presumably serves to remove the mercurial residues from the protein (19). Recon- stitution of trichloracetic acid-precipitated protein can also be effected in the same way, although in this instance the thiol agent is presumed to reduce disulfide bonds produced by oxidation dur- ing the acid denaturation. The reconstitution of the urea-oxi- dant protein is unique to the extent that a substantial yield can be obtained in the absence of added sulfide. The rationale for this is clear in the light of the previous discussion, for the apoprotein

itself contains the original sulfide moiety and this is released by the dithiothreitol. Thus the dithiothreitol has a dual role in this system, the simultaneous release of sulfide and the genera- tion of free mercaptide groups.

The product of reconstitution of the urea-oxidant denatura- tion behaves as ferredoxin by all the criteria that we have used, namely optical, circular dichroism, and electron paramagnetic resonance spectroscopy, chemical analysis, and biological ac- tivity. If there are any differences between the native and reconstituted protein they are subtle.

The effects of pH and NaCl concentration on the rate of bleaching by oxygen in the presence of urea are similar to the effects of these parameters on the stability of the protein in the absence of urea. Increased pH and high NaCl concentration clearly stabilize the protein in urea-oxygen solution (Fig. 1). The products of Fe(CN)$- denaturation and oxygen-urea de- naturation appear to be similar, although in the latter case less labile sulfur is bound in the oxidized state (Tables IV, VI, and VII). The more important question of whether oxygen de- naturation of native protein and the urea-facilitated oxidative denaturation yield the same products has not been investigated with spinach ferredoxin. However, with putidaredoxin (Table VI), which is particularly labile to oxygen and quickly loses all of its visible color at room temperature unless stored in 0.01 M mer- captoethanol, one finds an identical sulfur-zero content for the spontaneously denatured protein and that produced by treating the protein (stored in mercaptoethanol) aerobically with urea at room temperature for 1 hour (Table VI).

A number of observations suggest that the oxygen denatura- tion is a complicated phenomenon. The loss of visible absorb- ance exhibits several phases and thus is qualitatively similar to that reported for adrenodoxin (14). Not all the labile sulfide be- comes bound to the protein during the reaction, and there is no obvious correlation between loss of visible absorption and loss of optical activity during denaturation.4 A more thorough study is in progress.

The oxygen sensitivity of spinach ferredoxin may serve as a prototype to rationalize a number of observations in the literature on the (oxygen) instability of these proteins. Thus, alfalfa (13) and parsley ferredoxins (20), the nitrogenase enzymes (21), and succinic dehydrogenase (22) are notoriously unstable in the pres- ence of air, as are the ferredoxins from M. Z~ct~Ziticus,~ C. pas- teurianum and Clostridium acidi-urici (23, 24). All of these pro- teins can be markedly stabilized by storage under nitrogen, and it seems reasonable that the origin of the instability is the oxida- tion of the mercaptide-sulfide system of these proteins. Indeed we have demonstrated bound sulfur-zero in several iron-sulfur proteins of bacterial, plant, and animal origin which were sub- jected to the urea-oxidant denaturation procedure. Xanthine oxidase, too, may be an example of this phenomenon. Thus, Stadie and Haugaard (25), and Mann and Quastel (26) have demonstrated sensitivity of this enzyme in crude tissue homog-

enates. In addition, putidaredoxin, as isolated, appears to be

partly denatured and the quality of this material can be improved by treatment with dithiothreitol (27). This is probably due to the sensitivity of the protein to oxygen as described above, with the concomitant reversal by the dithiol.

From a practical point of view these observations have an im-

4 D. Petering and G. Palmer, unpublished observations. 5 R. Matthews and G. Palmer, unpublished observations.

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Issue of February 10, 1971 D. Petering, J. A. Fee, and G. Palmer 653

portant consequence, one which has already been realized em- pirically (13, 24). It is that those proteins of this class which are unstable aerobically should be stored under Nz. A conveni- ent device for both the storage and dispensing of solutions from under a nitrogen atmosphere has been described elsewhere (20).

The rapidity of the oxidative denaturation of ferredoxin in

urea solutions by both oxygen and ferricyanide and the high yields of bound sulfur-zero resulting from the reaction suggest that we are dealing with a specific phenomenon which is a conse- quence of the unique properties of the active site of these pro- teins. This concept is strengthened by the failure of solutions

of cysteine and sulfide to yield significant amounts of cysteine trisulfide with a variety of oxidants.6 Thus, it seems very un-

likely that this is a reaction which occurs as a secondary conse- quence of releasing iron and labile sulfide into solution, suggesting that the oxidation reaction itself is the cause of the denaturation. Physical measurements on ferredoxin in anaerobic urea show that the iron site of this protein has been perturbed but not destroyed

(6). Hence oxygen and ferricyanide react with a system closely resembling that found in the native protein. Considerable in-

formation about this site could be gained if the amino acid resi- dues which contribute the outer sulfurs to the proposed tri- sulfide are specific and identifiable by chemical means.

Acknowledgments-We are greatly indebted to the many col-

leagues who provided us with valuable proteins and reagents. In particular we would like to express our appreciation to Dr. M. J. Hunter for her many courtesies and to Miss S. Fielek, Miss A. B. Harris, and Mr. J. Trojanowski for their very capable as-

sistance during various phases of this work.

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David Petering, James A. Fee and Graham PalmerTHE FORMATION OF PROTEIN-BOUND SULFUR-ZERO

The Oxygen Sensitivity of Spinach Ferredoxin and Other Iron-Sulfur Proteins:

1971, 246:643-653.J. Biol. Chem. 

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