[34] Cytochromes: Bacterial

344

COMPONENTS

[34]

b-559Hp is calculated on the assumption of a value of 20 cm~/mmole for the millimolar extinction coefficient. Cytochrome b-559Le and b-563: An osmotically shocked dilution of the chloroplast preparation is made as described for cytochrome b-559Hp, with the further addition of 1.25 mM hydroquinone and catalase (400 U/ml of suspension, i.e., 0.5 td crystalline suspension from Boehringer to each milliliter of suspension) to the suspension before it is divided between two 1-cm cuvettes. The base line is drawn with the spectrophotometer. A freshly prepared solution of sodium dithionite (for each milliliter of suspension, 10 ~l of a solution containing 50 mg/ml) is added with rapid mixing to one cuvette. The spectrum is scanned rapidly and repeatedly between 580 and about 530 nm, and the time after the addition of dithionite is noted each time the peak position is reached. Measurements are continued until no further increase is detectable (about 10 minutes, but the rate of reduction can be controlled by the amount of dithionite added). For each time point measurements are taken on the heights of the peak at 559 and 563 nm above the line drawn through the minima at either side of the peak. For each wavelength the log of the difference between the final peak height and the height at time t is plotted against time (Fig. 5). From the intercept the peak heights of the rapidly reduced b-559Lp and the slowly reduced b-563 are calculated. A millimolar extinction coefficient of 20 cm2/mmole is again assumed for each component.

[34] Cytochromes: Bacterial

By ROBERTG. BARTSCH As is indicated by the tabulation of presently known soluble tyrochromes of photosynthetic bacteria (see Table I), this group of organisms is a prolific source of diverse cytochromes. Many of the cytochromes have distinctive properties which make them interesting examples of the variety of environments the apoproteins can impose on hemes. The information summarized in the first six columns of Table I will aid in identifying those bacterial cytochromes presently known. With the aid of information presented in the last four columns of Table I, it should be possible to devise suitable chromatographic procedures to purify the cytochromes listed. In general, pI values less than 7 indicate that the cytochrome can be chromatographed on DEAF-cellulose; values greater

[34]

CYTOCHROMES: BACTERIAL

345

than 7 indicate basic cytochromes which are chromatographed on CM-cellulose.

Assay Methods Absorption spectra measurements are routinely used to assay the cytochromes. Because of the high concentration of strongly absorbing carotenoids and chlorophylls in wild strains of the photosynthetic bacteria, it is difficult to detect cytoehrome absorption bands with a hand spectroscope. In addition, the effect of light scattering makes it difficult to measure eytoehrome spectra of whole cell suspensions even with many sensitive speetrophotometers. Reduced-minus-oxidized difference spectra between two initially equivalent buffered samples must be resorted to for an indication of the cytochrome content of a suspension of cells or of membrane fragments. With a split-beam spectrophotometer such as a Cary 14 (or 15) instrument, one of the pair of samples may be left unaltered, chemically oxidized with a small amount of potassium ferricyanide or even sodium hypochlorite, or exposed to photoactive light to test for a light-induced reaction. The other sample may be reduced by endogenous reduetants, or by the requisite amount of chemical reduetants such as sueeinate, aseorbate, NADH, sodium dithionite, or thiol compounds such as 2-mereaptoethanol or dithiothreitol. Alternatively a dual wavelength speetrophotometer may be used in the manner described elsewhere in this series. 1 The same techniques are applicable to cell-free extracts and purified eytoehromes. Because of interference by chlorophyll and especially carotenoid pigments, because eytoehromes such as eytochrome cc' lack distinctive absorption peaks, or because the eytoehrome absorption peaks in a complex mixture overlap, it is essentially impractical to estimate accurately the amounts of the individual eytoehromes contained in whole cells, or even in most etude extracts. Relatively simple eytoehrome mixtures such as that in R. rubrum extracts are an exception (see Table III). A further complication is the failure of bound eytoehromes cc' and certain others to react with carbon monoxide,2 whereas the unbound forms react readily, with the consequence that the carbon monoxide derivatives of these eytochromes are not reliable indexes of their concentration in the cells or ehromatophores. The description of eytoehrome analysis procedures as applied to mitoehondrial systems by Rieske a is a useful example of techniques applicable to the bacteria. 1This series, Vol. XXIV. 2M. A. Cusanovieh and M. D. Kamen, Biochim. Biophys. Acta 153, 376 (1968). 3j. S. Rieske, this series, Volume 10 [76].

346

COMPONENTS

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[34]

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[34]

CYTOCHROMES ; BACTERIAl,

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II

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347

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[34]

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[34]

CYTOCHROMES : BACTERIAL

349

TABLE II ALKALINE PYRIDINE FERROHEMOCHROME]~XTINCTION COEFFICIENTS

Cytochrome

~nm

c b

550 ± 1 556 d: 1 587 4- 2

a

~mM

31.18a 34.7c 24e

A~mM,r e d - o x 19.1 b 30~ --

a T. Flatmark, unpublished value for pure ox heart cytochrome c, quoted by K. Dus, H. De Klerk, R. G. Bartsch, T. Horio, and M. D. Kamen, Proc. Nat. Acad. Sci. U.S. 57, 367 (1967). b H. Theorell, Biochem. Z. 285, 207 (1936). c K. G. Paul, H. Theorell, and A./~eson, Acta. Chem. Scan& 7, 1284 (1953). d See J. S. Rieske, this series, Volume X [76]. • W. A. Rawlinson and J. H. Hale, Biochem. J. 45, 247 (1949). In Table I are listed the available extinction coefficients for the cytochromes. Most of the values are determined as absorbance per mole of heme. The heme content of the proteins is estimated by measuring the alkaline pyridine ferrohemochrome spectra. For this purpose the protein is dissolved in a 3:1 mixture (v/v) of 0.2 M N a O H solution and pyridine (reagent grade), the absorption spectrum of the ferrihemochrome is measured, then several crystals of sodium dithionite are added to reduce the sample, and the ferrohemochrome spectrum is measured. The extinction coefficients used for the several types of cytochrome are listed in Table II. Alternatively, the reduced-minus-oxidized difference spectrum m a y be useful with samples such as whole cells which scatter light, although ordinarily most cells and proteins dissolve completely in the strongly alkaline solution. It is necessary to remove interfering carotenoid and chlorophyll pigments from cells or membrane preparations before preparing the alkaline pyridine hemochrome solutions. For this purpose the material is extracted and centrifuged several times in a centrifuge tube with acetonemethanol (7:2, v / v ) ~ until the residue is essentially free of the soluble pigments. The alkaline pyridine ferrohemochrome spectra give qualitative identification of the type of cytochrome in a preparation. The minimum requirement suggested ~ for classifying a cytochrome as to type is t o measure the alkaline pyridine ferrohemochrome spectrum of the cytochrome as well as of the heme derived from the protein. To extract dissociable heme from a cytochrome, the ice cold preparation is made p H 2 with HCI and is extracted with 2-3 col of cold acetone, where4 M. Florkin and E. Stotz, "Comprehensive Biochemistry," Vol. 13, p. 18. Elsevier, Amsterdam, 1963.

350

COMPONENTS

[34]

upon the protein is precipitated, or with 1-2 vol of ice-cold methylethylketone, which is immiscible with the aqueous phase. 5 The protoheme of cytochrome b, and the formyl heme of cytochrome a, are extracted into the organic phase, whereas the covalently bound mesoheme of cytochrome c remains with the protein in the aqueous phase. The organic phase may be evaporated and the residue examined directly in the alkaline pyridine solvent, or better, the heme is transferred to ethyl ether, and then is extracted into 0.1 M NaOH. Finally the alkaline pyridine ferrohemochrome spectrum is measured. The protein or aqueous residue is also tested for the alkaline pyridine ferrohemochrome spectrum of cytochrome c. The diagnostically useful absorption maxima for identifying the hemes are indicated in Table II. Once the heine group is identified, the cytochrome can generally be given its proper designation. Methods for Determining Useful Physical Properties Electrophoresis of proteins using the isoelectric focusing technique of Vesterberg and Svensson6 provides useful information about the charge properties, or isoelectric pH, as well as purity of the cytochromes. The technique is also useful as an analytical method for separating differently charged members of a group of cytochromes. For example, some seven or more cytoehromes cc' ranging between pI 4 and pI 9, and eight or more cytochromes c2 can be separated from a crude extract of R. r u b r u m cells by the isoelectric focusing method. 7,s The technique has been used on a small scale preparative basis to separate mixtures for amino acid analysis. The Ampholine (LKB) electrolytes required seem to bind tightly to the separated proteins, but these can be separated by precipitation of the protein by ammonium sulfate, which presumably displaces the electrolyte. The measurement of oxidation-reduction potentials of small cytochrome samples is most conveniently done by the method of mixtures as described by Davenport and HilP and by Velick and Strittmatter, 1° wherein the relative concentration of oxidized and reduced cytochrome is determined spectrophotometrically in the presence of different ratios of oxidized and reduced components of a redox buffer. The redox buffer 5F. W. J. Teale, Biochim. Biophys. Acta 35, 543 (1957). eO. Vesterberg and H. Svensson,Acta Chem. Scand. 20, 820 (1966). TK. Sletten and M. D. Kamen, in "Structure and Function of Cytochromes" (K. Okunuki, M. D. Kamen, and I. Sekuzu, eds.), p. 472. Univ. of Tokyo Press, Tokyo, 1968. 8R. G. Bartsch, T. Kakuno, T. Horio, and M. D. Kamen, submitted to J. Biol. Chem.

9H. E. Davenport and R. Hill, Proc. Roy. Soc. Ser. B 139, 327 (1952). ~*S. F. Velick and P. Strittmatter, J. Biol. Ch~m. 221, 265 (1956).

[34]

CYTOCHROMES : BACTERIAL

351

is at least 100-fold more concentrated than the eytoehrome and may consist of reagents such as ferri-ferrocyanide (Era,7 ~ 0.4 V),9 ferri-ferrooxalate (Em,7 ---- 0 V),11 or ferri-ferro-EDTA (Era,7 = 0.1 V).12 Alternatively, the initially oxidized cytochrome plus any of the above redox mediators, or various redox dyes of suitable potential range, may be titrated with 10 mM sodium dithionite in 0.1M Tris.HC1, pH 7.5-8.0, or other alkaline buffer. The Eh of the mixture may be measured vs. a Pt electrode, and the extent of reduction of the cytochrome is measured spectrophotometrically. In general, anaerobic conditions must be used to exclude interference by atmospheric oxygen in any of these titrations, although high redox potential systems such as cytochrome c: or ] may be titrated in open cuvettes with a ferri-ferrocyanide buffer with no noticeable effect by atmospheric 02. Molecular weights or, more accurately, relative cross-sectional sizes of the cytochromes are generally approximated by use of the Andrews 13 technique of comparing the relative rates of migration of the protein and protein standards through Sephadex G-75 or G-100 columns. Such approximate values can be used to determine the scale factor required to convert accurate amino acid analysis values to an accurate formula weight of the heme protein. If the heme content and formula weight are determined with aliquots of the same sample, the number of hemes per molecule can be derived, 14 or at least the number of hemes per minimum integral number of amino acid residues can be determined. General Purification Procedures The purification of most of the cytochromes listed in Table I is carried out in a standardized manner as illustrated in the schematic flow sheet (Fig. 1). For desalting extracts or eytochrome solutions, it is convenient to use a Sephadex G-25 column equilibrated vs. distilled water or a low concentration of buffer suitable for starting chromatographs on D E A E - or CM-cellulose. The conductivity of the effluent from Sephadex G-25 desalting columns is conveniently monitored with a flow cell and conductivity meter such as the Radiometer CDM. Customarily, the average conductivity of the desalted solution is kept below one-third that of the initial column equilibration buffer, and for this purpose the sample size is kept somewhat less than one-third the Sephadex G-25 R. Hill, Nature (London) 174, 501 (1954). G. Schwarzenbach and J. Heller, Helv. Chim. Acta 34, 576 (1951). P. Andrews, Biochem. J. 91, 222 (1964). ~4K. Dus, It. De Klerk, R. G. Bartsch, T. Horio, and M. D. Kamen, Proc. Nat. Acad. Sci. U.S. 57, 367 (1967).

Cell suspension (100 g wet wt in 400 ml of 0.2 M I

I

Precipitate cellular debris

Tris.HC1, pH 7.3-8.0) Break cells with Sorvall-Ribi cell fractionator, 20,000 psi, 20 ° Centrifuge 10-30 minutes at 30,000 g

I

Cell-free extract I Centrifuge 2-3 hours at 100,000 g

I

Precipitate chromatophores

Supernatant solutions DEAE-cellulose, Selectacel Standard, 75-ml bed volume, -[-0.2 M buffer Wash with 400 ml of application buffer

I

Unadsorbed proteins Desalt, Sephadex G-25 DEAE-cellulose, type 20, 100-ml bed volume, +1-10 mM Tris-HC1, pH 8.0 Wash with 200 ml of application buffer

Elute with 0.3-0.4 M NaC1 in 20 mM Tris.HC1, pH 7.3 Ferredoxin

Elute cytochrome zones with increasing concentration of NaC1 in buffer Desalt each zone, Sephadex G-25 Concentrate on DEAE-cellulose, Type 20, 3-ml bed volume, elute with 0.5 M NaC1 in 20 mM buffer Fractionate with ammonium sulfate a Desalt and chromatograph on DEAE-cellulose columns Chromatograph on Sephadex G-100 Concentrate Crystallize from ammonium sulfate solution Acidic cytochromes

Unadsorbed basic proteins CM-ceUulose, Selectacel standard, 30-50 ml bed volume, ~ 1 mM phosphate, pH 5.0-6.0 Wash with 200 ml application buffer Elute colored bands with increasing concentrations of buffer Desalt each zone, Sephadex G-25 or Bio-Gel P-2 Concentrate on CM-cellulose, standard, 3-ml bed volume, elute with 0.1 or 0.2 M P~, pH 7.0 Fractionate with ammonium sulfatea Chromatograph on Sephadex G-100 Concentrate Crystallize Basic cytochromes

I

Fro. 1. General purification scheme used for bacterial cytochromes. See text for details. a A. A. Green and W. L. Hughes, this series, Volume 1 [10]. 352

[34]

CYTOCHROMES: BACTERIAL

353

column bed volume. Some crude extracts stain the columns with colored membrane fragments which can generally be removed by eluting the column with Triton X-100 in 10 mM NaOH (1%, v/v). The general procedure for use of D E A E - and CM-cellulose chromatography columns described by Peterson and Sober 1~ is followed. The CM-cellulose and DEAE-cellulose Selectacel-Standard, a rapid flow grade, and the DEAE-cellulose Selectacel Type 20, a moderate flow grade, are obtained from the Brown Co., Berlin, New Hampshire. To regenerate DEAE-cellulose columns it is useful to include Triton X-100 in 0.2M NaOH (1%, v/v) plus 0.5M NaC1 to elute colored membrane fragments which otherwise adhere irreversibly to the adsorbent. Several nearly standardized preparations are given here to illustrate the general scheme. For this purpose the descriptions are scaled for 100 g wet weight of cells.

Rhodospirillum rubrum Cytochromes The simplest cytochrome pattern encountered is that of Rhodospirillure rubrum, from which only cytochrome c~ and cytochrome cc' with numerous isocytochrome forms ~,s and cytochrome bs~s,8 have been isolated. Cytochrome c~ (pI : 6.2) and cytochrome cc' (pI = 5.6) predominate, to the extent of approximately one-half of the total content of the two cytochromes in crude extracts. A small part of the same predominant cytochromes have been tentatively identified as the tightly bound chromatophore cytochromes which can be shown to undergo lightinduced oxidation-reduction reactions. 1~ The three cytochromes are acidic and consequently are chromatographed on DEAE-cellulose. The purification of the two cytochromes c is summarized in Table III. For none of the cytochromes discussed in this article is there available a complete set of yield and purity values per purification step to make such a table complete. Cytochrome b~8. To separate conveniently cytochrome b~s, the crude eluate from the first DEAE-cellulose column used to remove ferredoxin is fractionated with ammonium sulfate. Cytochrome b~s is precipitated in the range 15-30% ammonium sulfate saturation, together with an FMN protein, the bulk of which remains in solution. The precipitate is dissolved in 40 ml of 0.1 M Tris-HC1, pH 8.0, and centrifuged for 1012 hours at 100,000 g. The precipitate of cytochrome b is dissolved in 10 ml of 0.1 M Tris.HC1, pH 8.0, and centrifuged at 30,000 g for 1 hour to remove insoluble material. The supernatant solution is adjusted to pH 4.6 by addition of 1 M acetic acid at 0 ° and immediately centrifuged is E. A. Peterson and It. A. Sober, this series, Volume V [1]. 1,T. Kakuno, R. G. Bartsch, K. Nishikawa, and T. Horio, submitted to J. Biochem. (Japan).

354

co~eoN~.NTS

[34]

TABLE III I~URIFICATION OF

Rhodospirillum rubrum CYTOCHROMES

Cytochrome c,a

Cytochrome cc 'b

yield

yield

yield

(~moles)

(~moles)

(AA,~5)

4.7 2.3 1.4

2.2 0.86 0.6

77

Crude extract 1st DEAE-cellulose 1st Crystallization

Cytochromeb6~¢

a AA550.m correctedfor 5A 550nmdue to cytochromecc'. AEmM. ~50(~2)= 22, 5e~M. ~50eo') = 25. bAA.8 a m , ~ e m M , 638 nm 4. c AA4~5 ,m, Na~S2O4-- AA426 nm, NADH. A t p H 7, 1 raM NADH c o m p l e t e l y reduces cytoehromes c~ and cc' in the crude extract. =

at 30,000 g for 10 minutes to remove unwanted colorless protein. The supernatant solution is immediately neutralized with 1 M sodium hydroxide. The clear solution is chromatographed on a Sephadex G-100 column (1 liter bed volume per 20-30 ml of solution) equilibrated with 1 M NaC1 in 0.1 M phosphate buffer, pH 7.0, in the cold room. The cytochrome is eluted in the void volume, followed by the remaining F M N protein. The best cytochrome bs~s fractions are combined, desalted with the aid of a Sephadex G-25 column, equilibrated with 1 mM phosphate buffer, pH 7.0, and then chromatographed on a DEAE-eellulose column (type 20, 30 ml bed volume) equilibrated with the same buffer. The cytochrome is eluted with 10 mM phosphate buffer, pH 7.0. Finally, the best fractions are pooled and precipitated with ammonium sulfate, over the range 15-20% saturation. Cytochromes c2 a n d cc'. Cytochromes c2 and c e can conveniently be precipitated from the ammonium sulfate supernatant solution remaining from the cytochrome b preparation by saturating the solution with ammonium sulfate. Residual cytochrome c2 remaining in solution can be concentrated by filtering the ammonium sulfate solution through a column of DEAE-cellulose (6-ml bed volume) ; this serves as a convenient filter aid which is superior to the more conventional types tried. The accumulated protein can be eluted off the column with water or 0.1 M Tris.HC1, pH 7.3. The precipitated proteins are dissolved in sufficient 0.1 M Tris.HC1, pH 8.0, to reduce the residual ammonium sulfate concentration to less than 20% saturation. The solution is desalted with Sephadex G-25 equilibrated with 1 mM Tris.HC1, pH 8.0. Alternatively, the crude ferredoxin-free extract may be desalted directly if no cytochrome b is to be recovered. The protein solution is then chromatographed at room temperature on a DEAE-cellulose column (type 20,

[34]

CYTOCHROMES : BACTERIAL

355

100-ml bed volume) equilibrated with 1 mM Tris.HC1, pH 8.0. After charging with the protein solution, the column is washed with 300 ml of 1 mM Tris.HC1, pH 8.0, and then with 300 ml of 10 mM Tris.HC1, pH 8.0, to distribute the protein in the column. Cytochrome v2 is then eluted with 400 ml of 30 mM Tris.HC1, pH 8.0, and the cytochrome cc' is eluted with 500 ml of 50 mM Tris.HC1, pH 8.0. There remains on the column as much as one-third of the total cytochrome which consists of the more acidic isocytochromes c:2 and cc' found in R. rubrum extracts. These isocytochromes may be eluted with 0.5 M NaC1 in 20 mM Tris. HC1, pH 8.0; after desalting with Sephadex G-25, they are best separated by electrophoresis with the isoelectric focusing technique. 8 The best fractions of cytochrome c2 in the 30 mM Tris.HC1 eluate are pooled, desalted with Sephadex G-25, concentrated on a DEAF--cellulose column (type 20, 2-ml bed volume) equilibrated with 1 mM Tris.HC1, pH 8.0, and eluted in concentrated solution with 0.5 M NaC1 in 20 mM Tris. HC1, pH 8.O. The best cytochrome cc' fractions are combined, desalted, concentrated on a DEAE-cellulose column (type 20, 2-ml bed volume) and eluted with the strong salt solution. Primarily to remove slowly migrating residual cytochrome c2, the cytochrome cc' is next chromatographed on a Sephadex G-100 column, equilibrated with 0.2 M NaC1 in 20 mM Tris.HC1, pH 7.3, in the cold. Again, the best fractions of cytochrome cc' are combined, desalted, and concentrated. Both concentrated cytochromes are next precipitated with ammonium sulfate, between 70 and 100% saturation. Finally the two cytochromes are induced to crystallize at room temperature from ~60% saturated ammonium sulfate in 0.1M Tris.HC1, pH 8.0. By three successive crystallizations of the main crops of each cytochrome, the predominant forms, isocytochrome c, pI 6.2, and isocytochrome cc', pI 5.6, can be isolated in pure form. Properties o] R. rubrum Cytochromes. The characterization of cytochrome bs~s is incomplete. One peculiarity to note is the extreme slowness with which the cytochrome becomes reduced, even with sufficient excess of sodium dithionite as reducing agent to make the reaction mixture completely anaerobic. Several hours may be required for complete reduction to occur in a spectrophotometer cuvette. The cytochromes c~ and cc' serve as the type examples for these two classes of cytochrome, and they have been extensively characterized (see Table I and indicated references). The amino acid sequence of cytochrome c2 has been reported. 17 1~K. Dus, K. Sletten, and M. D. Kamen, J. Biol. Chem. 243, 5507 (1968).

356

COMPON~.~TS

[34]

Chromatium Cytochromes

At least three cytochromes can be isolated from extracts of Chrostrain D. The extract free from ferredoxin is desalted on a Sephadex G-25 column, made 2 mM with respect to Tris.HC1, pH 8.0, and chromatographed at 4 ° on a DEAE-cellulose column (Type 20, 100-ml bed volume) equilibrated with the same buffer. After preliminary washing with 400 ml of the application buffer to distribute the proteins through the column, the small amount of soluble cytochrome c~5~¢~5~) ( C h r o m a t i u m cytochrome ]) is eluted with 2 mM Tris.HC1, pH 8.0. Some more of the cytochrome may be eluted together with oxidized high potential iron protein (HiPIP) with 400 ml of 20 mM NaC1 in 20 mM Tris.HC1, pH 8.0. Reduced H i P I P is eluted with 400 ml of 40 mM NaC1 in 20 mM Tris.HC1, pH 8.0. Next, an as yet uncharacterized cytochrome c~51 may be eluted with 400 ml of 60-80 mM NaCI in the buffer. Cytochrome cc' is eluted with 80-100 mM NaC1 in the buffer, followed by cytochrome c552 eluted with 400 of ml 0.14-0.18M NaC1 plus buffer. A severalfold greater yield of cytochrome c553(5~o) can be extracted from cells, or the crude chromatophores from the 100,000 g centrifugation, by extracting twice with 50% acetone plus cell or particle suspension in. 10 mM Tris.HC1, pH 7.3, in the coldJ s The chilled acetone (--10 °) is rapidly stirred into the buffered chromatophore suspension in the cold, and then left for 10 minutes. The suspension is centrifuged for 10 minutes at 10,000 g at - - 2 0 °. The residue is resuspended in 10 mM Tris.HC1, pH 7.3, and again extracted with 50% cold acetone. The supernatant solutions are decanted and poured immediately through a DEAE-cellulose column (type 20, 50-ml bed volume) equilibrated with 10 mM Tris.HC1, pH 7.3. A small red-colored zone of the cytochrome accumulates ahead of a broad brown-colored zone consisting of H i P I P plus chromatophore fragments. If whole cells are extracted, ferredoxin also accumulates at the very top of the column. The cytochrome is eluted from the column with 20 mM Tris.HC1, pH 7.3. The cytochrome c~53(55~) fractions are desalted with Sephadex G-25, adjusted to 1 mM Tris.HC1, pH 8.0, and chromatographed again on a DEAE-cellulose column (type 20, 20 ml bed volume) in the manner described. The best fractions (A2Ts/A4~7. reo ~ 0.12) are pooled, desalted, and concentrated with the aid of a small DEAE-cellulose column. Both the cytochrome cc' and cytochrome c5~2 fractions are desalted on a Sephadex G-25 column, adjusted to pH 8.0, adsorbed on D E A E cellulose columns and eluted in concentrated solution with 0.5 M NaC1 matium

la M. A. Cusanovich and R. G. Bartsch, Biochim. Biophys. Acta 189, 245 (1969).

[34]

CYTOCHROMES: BACTERIAL

357

plus 20 mM Tris-HC1, pH 7.3. The concentrated solutions are fractionated with ammonium sulfate. The precipitate formed between 50 and 100% saturated ammonium sulfate is dissolved in 0.1 M Tris.HC1, pH 7.3. The cytochrome cc' fraction is desalted with a Sephadex G-25 column, the solution adjusted to pH 8.0 and adsorbed on a DEAE-cellulose column (type 20, 30-ml bed volume) equilibrated with 20 mM TrisHC1, pH 8.0. After preliminary washing with 400 ml of 20 mM Tris.HCl, pH 8.0, followed by 300 ml of 60 mM NaC1 in 20 mM Tris.HC1, pH 8.0, the cytochrome zone is eluted with 500 ml of 80-100 mM NaC1 in the buffer. The best fractions (purity index A~s4/A39~ = 0.4-0.5) are pooled, desalted, and concentrated with the aid of a small DEAE-cellulose column. Finally, the concentrated solution is fractionated with ammonium sulfate, the main fraction of the cytochrome precipitates between 70 and 100% saturation. The precipitate is dissolved in 0.1 M Tris.HC1, pH 7.3. The sample is essentially homogeneous if A28~/ A39o,o~ ~ 0.33 is attained, repetition of the fractionation with ammonium sulfate may be needed to reach this purity level. The cytochrome has never been crystallized. The cytochrome c5~2 fraction precipitated by ammonium sulfate is dissolved in 0.1 M Tris.HC1, pH 7.3, and chromatographed on a Sephadex G-100 column (l-liter bed volume per 20-30 ml solution) equilibrated with 0.2 M NaC1 in 20 mM Tris.HC1, pH 7.3. The first colored fraction consists of membrane fragments in the void volume followed by a flavoprotein with dye reductase capabilities. 19 Cytochrome c5~2 is eluted next, followed by residual cytochrome cc'. The best fractions of cytochrome c ~ are pooled, desalted with a Sephadex G-25 column, and concentrated with the aid of a DEAE-cellulose column. Dependent on the purity achieved, the sample may be chromatographed again on DEAE-cellulose (type 20, 30-ml bed volume) in the manner described for cytochrome cc', with the difference that the column is first eluted with 300 ml of O.12M NaC1 in 20 mM Tris-HC1, pH 8.0, and then with 0.16M NaC1 in the same buffer to elute the cytochrome. The best fractions are pooled, desalted and concentrated. The cytochrome prepared this way, or possibly the best fraction from the Sephadex G-100 cbromatogram, is subjected to stepwise ammonimn sulfate fractionation over the range 60-90% saturation, in 10% saturation increments. The best fraction may crystallize upon standing ia ammonium sulfate solution of the same or slightly lower concentration from which it was precipitated, generally 60-70% ~9M. A. Cusanovich, R. G. Bartsch, and M. D. Kamen, Biochim. Biophys. Acta 153, 397 (1968).

358

COMPONENTS

[34]

saturation. For the essentially homogeneous cytochrome the purity index, A2so/A41o, ox = 0.55.

The flavin of this cytochrome slowly dissociates throughout the purification. Consequently, in part, the purification procedure eliminates denatured flavin-free heme protein. The absorption ratio, A4~o/A52o, for the oxidized protein is used as a measure of the relative amounts of flavin and heme of the preparation, for the best samples the ratio ~--1.3. The flavin can be dissociated from the cytochrome by incubation at pH > 9, with saturated urea or 4 M guanadine hydrochloride, or with a mercurial such as p-chloromercuribenzoate/° Properties o] the Chromatium Cytochromes. Cytochrome c553~5~o~is a nonautoxidizable eytochrome, (Em,~ = +0.32V) which is isolated predominantly in the reduced state. The purified protein appears to polymerize upon storage, inasmuch as molecular weights ranging from 13,000 to 50,000 daltons are found by sedimentation-equilibrium analysis. The cytochrome resembles algal cytochromes ] in spectroscopic and physical properties, and may be the soluble form of the high-potential chromatophore-bound cytochrome c5~5 which undergoes light-induced oxidation in chromatophores and whole cells. Cytochromes cc' and c~2 are autoxidizable and are isolated in the oxidized state. On the basis of similar absorption spectra and redox potentials, cytochrome c~2 has been tentatively identified with cytochrome c4~3.5 which undergoes light-induced oxidation in anaerobic whole cells 21 or in chromatophores adjusted to Eh < 0 V. 22 The same cytochrome in starved cells responds to a reducing substrate such as sodium thiosulfate, and therefore has been implicated in a noncyclic electron transfer system.23 Both of the isolated autoxidizable cytochromes combine with carbon monoxide, but the bound forms in the chromatophore fail to react with the ligand. 2 The bound cytochromes can be solubilized from an acetone powder of the particles or by extracting the particles with 2% Triton X-100 in 0.1 M Tris-HC1, pH 7.3. Upon purification by the above procedure, the cytochromes have properties identical with the proteins isolated from the aqueous extract. R. G. Bartsch, T. E. Meyer, and A. B. Robinson, in "Structure and Function of Cytochromes" (K. Okunuki, M. D. Kamen, and I. Sekuzu, eds.), p. 472. Univ. of Tokyo Press, Tokyo, 1968. ~ J. M. 01son and B. Chance, Arch. Biochem. Biophys. 88, 26 (1960). ~ M. A. Cusanovich, R. G. Bartsch, and J. M. Olson, in "Comparative Biochemistry and Biophysics of Photosynthesis" (K. Shibata, A. Takamiya, A. T. Jagendorf, and R. C. Fuller, eds.), p. 186. Univ. of Tokyo Press, Tokyo, 1968. 25S. Morita, M. Edwards, and J. Gibson, Biochim. Biophys. Acta 109, 45 (1965).

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Rhodopseudomonas palustris Cytochromes Rhodopseudomonas palustris (van Niel strain No. 2.1.37) provides the most complex assortment of cytochromes encountered among the photosynthetic bacteria so far examined. Two acidic cytochromes plus five cytochromes with basic isoelectric pH values can be isolated from the organismY 4,2~ Except for cytochromes c2 and c', the following directions are not optimized for recovery of these 7 cytochromes. The extraction procedure and ferredoxin removal method already described are used. The ferredoxin-free extract is desalted on a Sephadex G-25 column equilibrated with 1 mM Tris.HC1, pH 8.0. The solution is next passed into a DEAE-cellulose column (Selectacel-Standard, 270-ml bed volume), equilibrated with 1 mM Tris.HC1, pH 8.0. The column is washed with 500 ml of the application buffer to stabilize the protein zones and to wash off unadsorbed proteins (eluate 1). The column is next washed with 500 ml of 0.1 M Tris.HC1, pH 8.0, to remove a mixture of cytochromes and dehydrogenases (eluate 2). The column is then eluted with 500 ml of 0.2 M NaC1 in 20 mM Tris.HC1, pH 8.0, to remove a mixture of a cytochrome b, an iron protein with an absorption spectrum like that of adrenal redoxin and a possible N A D P H dehydrogenase 2~ (eluate 3). Eluates 2 and 3 are thereby considerably concentrated relative to the starting extract and the basic proteins are separated in eluate 1. Eluate 1 is adjusted to pH 5.5, either by careful titration with 1 M acetic acid, or by addition of small increments of Dowex 50-H ÷ ion exchange resin, which is filtered off once the desired pH is reached. With the latter method, the ionic strength of the solution is kept at a minimum. The solution is made 100/~M in 2-mercaptoethanol to reduce high potential cytochromes and is then chromatographed on a CM-cellulose (Selectacel-Standard, 30-ml packed volume) equilibrated with 1 mM phosphate buffer, pH 6.0. The column is washed with 400 ml of the application buffer to develop the clearly separated colored bands and then the following cytochromes are eluted with 400-500 ml portions of the buffer indicated: cytochrome c~55 (2 mM phosphate buffer, pH 6.0), cytochrome c~1.5 plus cytochrome c' (5 mM phosphate buffer, pH 6.0), eytochrome c~.... (20 mM phosphate buffer, pH 6.0), and cytochrome c5~4 (50 mM phosphate buffer, pH 6.0). Each cytochrome zone is separately desalted by passage through a Sephadex G-25 column equilibrated with 1 mM phosphate buffer, pH 5.5, and concentrated with the aid of small (3-ml packed volume) CM-cellulose columns. With the exception of the cyto-

~ R. G. Bartsch, T. Horio, and M. D. Kamen, submitted to Biochim. Biophys. Acta. R. G. Bartsch and T. E. Meyer, private communication, 1970. T. Yamanaka a~ndM. D. Kamen, Biochim. Biophys. Acta 131, 317 (1967).

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chrome c' fraction, the proteins are eluted with 0.1 M phosphate buffer, pH 7.0. The cytochrome c' solution is made 100 pit/ with 2-mercaptoethanol and adsorbed on a concentration column. The contaminating cytochromes c555 and c5~1.5 are reduced by the 2-mercaptoethanol and therefore are easily eluted with 2 mM phosphate buffer, pH 5.5, leaving the more positively charged, oxidized cytochrome c' adsorbed. The latter cytochrome is eluted with 5 mM phosphate buffer, pH 5.5. The now separated fractions are concentrated in the above manner. A recurrent problem with highly basic cytochromes is the obvious loss of a portion of the colored proteins by adsorption on the Sephadex G-25 column. This loss is not completely eliminated by the use of the low concentration of buffer required for subsequent adsorption on C M cellulose columns. Freshly hydrated Bio-Gel P-2 desalting columns do not initially adsorb the cytochromes, but slowly deteriorate with use and eventually behave like Sephadex G-25. The concentrated cytochrome c' is precipitated over the range 7090% saturated ammonium sulfate. The precipitate is dissolved in 3 ml of 0.1 M Tris.HC1, pH 8.0, solid ammonium sulfate is added to --70% saturation and the cytochrome crystallizes after several days standing at room temperature. Cytochrome c2 is passed through a Dowex-2 anion exchange column (2-ml bed volume) charged to two-thirds capacity with ferricyanide. The solution of completely oxidized cytochrome is then desalted by passage through a Bio-Gel P-2 column equilibrated with 1 mM phosphate buffer, pH 6.0. The desalted solution is then chromatographed on a C M cellulose column (Selectacel-Standard, 40-ml bed volume) equilibrated with 1 mM phosphate buffer, pH 6.0. The main zone of cytochrome c2 is eluted with 500 ml of 25 mM phosphate buffer, pH 6.0, desalted with the Bio-Gel P-2 column, concentrated on a small CM-cellulose column and eluted with 1 M NaC1 in 0.1 M Tris.HC1, pH 8.0. The cytochrome is precipitated between 65 and 90% saturated ammonium sulfate, then dissolved in 3 ml of 0.1M Tris.HC1, pH 8.0, and reprecipitated in the same manner. The precipitate is dissolved in 0.1 M Tris.HC1, pH 8.0, containing 20 mM 2-mercaptoethanol to reduce all the cytochrome. Sufficient solid ammonium sulfate is added to the solution at room temperature until slight turbidity appears (at ~ 7 0 % saturation). After 10 minutes standing the solution is centrifuged at 20 ° for 5 minutes at 30,000 g to remove the precipitate. Upon further standing fine pink crystals develop somewhat more rapidly than does a gelatinous precipitate. The early crop of crystals is collected before much gelatinous material accumulates. Recrystallization three or four times in succession in the

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same manner gives --20% recovery of cytochrome c2, free of the amorphous material. Thus far no other technique for removing the latter material has been devised. The three remaining concentrated basic cytochromes are precipitated with ammonium sulfate: cytochrome c554 (50-70% saturation), cytochrome c5~1.5 (60-80% saturation) to cytochrome c5~5 (80-100% saturation). Each precipitate is dissolved in 3 ml of 1 M phosphate buffer, pH 7.0, and chromatographed on a Sephadex G-75 column equilibrated with 0.1 M NaC1 in l0 mM phosphate buffer, pH 7.0, to remove colorless impurities. The best cytochrome fractions are pooled, desalted and concentrated with small CM-cellulose columns. Small quantities of nearly homogeneous cytochromes are obtained. Eluate 2 is cooled to 0 ° and adjusted to pH 5 with 2 M acetic acid. The suspension is centrifuged at 30,000 g for 10 minutes to remove the bulky precipitate. Occasionally a portion of the cytochrome c552 is precipitated and may be extracted from the bulk of denatured precipitate with 0.1 M Tris.HC1, pH 8.0. The supernatant solution is immediately adjusted to pH 6.0. Any precipitate which forms is removed by centrifugation. The solution is then desalted with Sephadex G-25 equilibrated with 1 mM Tris-HC1, pH 8.0, and chromatographed at room temperature on a DEAE-cellulose column (type 20, 55-ml bed volume) equilibrated with the same buffer. Cytochrome c~2 (c3) is eluted with 450 ml of 2 mM Tris.HC1, pH 8.0. Two NADH-dehydrogenases contaminated with cytochrome c~5., may subsequently be eluted. 24 As an alternative procedure the eluate 2 is precipitated with saturated ammonium sulfate, the precipitate is dissolved in 0.1M Tris.HC1, pH 7.3 and the solution is chromatographed on a Sephadex G-100 column (l-liter bed volume for each 20-30 ml of concentrated solution) equilibrated with 0.2M NaC1 in 20 mM 'Iris.HC1, pH 7.3, in the cold. Presumed membrane fragments are eluted in the void volume, followed by the flavin-containing dehydrogenases and finally cytochrome c~5~. The cytochrome c552 fraction is desalted with Sephadex G-25 and concentrated with a small DEAE-cellulose column. The concentrated solution is fractionated with 80-100% saturated ammonium sulfate to precipitate the cytochrome. The precipitate is dissolved in 0.1M phosphate buffer, 7.0, and chromatographed on a Sephadex G-75 column to eliminate colorless impurities. The best fractions are pooled, desalted, and concentrated with a small DEAE-cellulose column. Even the best preparation made to date may not be homogeneous. Although certainly present in eluate 3, cytochrome b~8 has not been successfully purified from extracts of R. palustris strain 2.1.37. How-

362

COMPONENTS

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ever, an undesignated strain of R. palustris isolated as a contaminant from a culture of Chlorobium thiosul]atophilum yields the cytochrome? ~ Eluate 3 from this organism is chilled in an ice bath, and 2 M acetic acid is added to adjust the solution to pH 5. The precipitate formed is removed by centrifugation, and the supernatantsolution is adjusted to pH 8.0 by cautious addition of 2M NaOH and then is desalted with a Sephadex G-25 column. The solution is next chromatographed on a DEAE-cellulose column (type 20, 20-ml bed volume) equilibrated with 0.1 M Tris.HC1, pH 7.3. After a preliminary wash with 200 ml of 0.1 M Tris.HC1, pH 7.3, to remove flavoproteins, an iron protein with the plant ferredoxin spectrum followed by cytochrome b~.~ are eluted with 0.14M NaCl in 20 mM Tris.HC1, pH 7.3. The cytochrome is desalted with a Sephadex G-25 column and concentrated wi~h the aid of a small D E A E cellulose column. The concentrated solution is then chromatographed on a Sephadex G-100 column (l-liter bed volume per 20-30 ml of solution) equilibrated with 0.2M NaC1 in 20 mM Tris.HC1, pH 7.3. The cytochrome is eluted slightly behind the void volume, followed immediately by the remaining iron protein. Again the best fractions of cytochrome b558 are pooled, desalted, and concentrated with a small DEAE-cellulose column. Solid ammonium sulfate is added to the concentrated cytochrome solution to ~ 3 0 % saturation. After standing in the cold for 5-10 hours, deep-red crystals form and continue to grow over a period of several days. However, the bulk of the cytochrome crystallizes within 24 hours and is collected by centrifugation before much amorphous colorless material accumulates. Upon recrystallization the purity index, A28o/ A419,ox = 1.3, is attained. The cytochrome is slowly reduced with sodium dithionite, several hours are required for a sample in an open cuvette to become completely reduced after addition of excess reductant. Properties o] the R. palustris Cytochromes. The available information about the properties of the R. palustris cytochromes is summarized in Table I. As yet no studies on the biochemical role of any of the cytochromes has been reported. Cytochromes c2 and cc' are the most abundant cytochromes c isolated, the others are recovered in no more than one-tenth the yield of those two.

Chloropseudornonas ethylicum Cytochromes An exception to the general methodology already described is offered by Chloropseudomonas ethylicum. An unknown component of the extracts prevents complete adsorption from a crude extract of cytochrome c551.5 to DEAE-cellulose, although the cytochrome c~5 can be successfully chromatographed (see Table I). Fractionation with ammonium

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BACTERIAL

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sulfate, either by eluting a total precipitate with an inverse ammonium sulfate gradient, 27 or by precipitating the crude extract between 40 and 100% saturated ammonium sulfate before chromatographing on a D E A E cellulose column 2s removes the interfering factor. After desalting with a Sephadex G-25 column, cytochrome c551.~ is chromatographed on a DEAE-cellulose column (type 20, 20-ml bed volume) equilibrated with 20 m M Tris. HC1, pH 7.3. Elution of the cytochrome is accomplished with 0 . 2 M NaC1 in the buffer, after preliminary elution of the column with 0 . 1 M NaC1 in the buffer. Both cytochromes are further purified by chromatography on a Sephadex G-100 column in the manner described for R. palustris cytochrome b. Finally the cytochromes are desalted and concentrated on DEAF-cellulose columns (type 20, 3-ml bed volume) and eluted with 0.5 M NaC1 in 20 m M Tris.HC1, pH 7.3. Cytochromes from Dark-Aerobic Bacteria From R. rubrum grown aerobically in the dark, cytochrome c2 has been prepared essentially indistinguishable from that of light-anaerobic cells. The cytochrome cc' content of the aerobic cells was negligible. A membrane-bound cytochrome b with properties like those of cytochrome o was detected, 29 but was not solubilized. A different pattern was found in one strain of R. spheroides2 °,31 In aerobic cells there is produced a membranebound cytochrome c~51 different from the anaerobic cytochrome c2, and the content of cytochrome c553 (probably equal to cytochrome c5~ in Table I) is greatly increased2 ° A short-term (18 hours) aerobic culture of the R. spheroides strain develops a cytochrome a, which can be solubilized and shown to function as an oxidase2 ~ The cytochrome c ~ , as well as a cytochrome b, are components of the solubilized preparation. Upon longer incubation, the cytochrome a disappears and a cytochrome o appears to act as an oxidase. 31

27j. M. Olson and E. K. Shaw, Photosynthetica 3, 288 (1969). 28T. E. Meyer, Ph.D. Thesis, Univ. of California, San Diego, 1970. 29S. Taniguchi and M. D. Kamen, Biochim. Biophys. Acta 96, 395 (1965). Y. Motokawa and G. Kikuchi, Biochim. Biophys. Acta 120, 274 (1966). ~ G. Kikuchi and Y. Motokawa, in "Structure and Function of Cytochromes" (K. Okunuki, M. D. Kamen, and I. Sekuzu, eds.), p. 174. Univ. of Tokyo Press, Tokyo, 1968.