characterization of purified cytochrome c1 from rhodobacter sphaeroides r-26

Biochimiea et Biophysica Acta 852 (1986) 203-211 203 Elsevier

BBA 42154

Characterization of purified cytochrome c~ from Rhodobacter sphaeroides R-26

Linda Yu, Jian-Hua Dong and Chang-An Yu Department of Biochemistry, Oklahoma State University, Stillwater, OK 74078 (U.S.A.)

(Received 16 April 1986)

Key words: Cytochrome cl; Electron transport; Bacterial photosynthesis; (Rb. sphaeroides)

Cytochrome c I from a photosynthetic bacterium R h o d o b a c t e r sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmoi heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome cl, but the amino acid composition and circular dichroism spectral characteristics are different. The berne moiety of cytochrome c ! is more exposed than is that of mammalian cytochrome cl, but less exposed than that of cytochrome c z. Ferricytochrome c 1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuripbenylsulfonate is added to ferricytochrome c t, suggesting that the sulfhydryl groups of cytoehrome c I are the electron donors for photoreduction. Purified cytochrome c l contains 3 5:0.1 moi of the p-chloromercuripbenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome ct, the bacterial protein does not form a stable complex with cytoehrome c z or with mammalian cytoehrome c at low ionic strength. Electron transfer between bacterial ferrocytoehrome c t and bacterial ferricytochrome cz, and between bacterial ferrocytochrome c I and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c t is calculated to be 228 mV, which is identical to that of mammalian cytochrome c v

The role of cytochrome c 1 in the mitochondrial electron-transfer system has been thoroughly documented since its discovery in the early 1940's [1,2]. Although the spectral similarity between cytochromes c~ and c made studies of cytochrome c 1 using intact organdies very difficult, much in- formation on mammalian cytochrome c x has been acquired through studies using purified preparations [3-9]. Although the electron-transfer system

Abbreviation: PCMPS, p-chloromercuriphenylsulphonic acid.

Correspondence: Dr. Chang-An Yu, Department of Biochem- istry, Okl',daoma State University, Stillwater, OK 74078, U.S.A.

of photosynthetic bacteria is comparable to that of mitochondria, participation of cytochrome c I in the photosynthetic cyclic electron transfer was not recognized until recently [10,11]. The observation of the redox heterogeneity of cytochrome c 2 in cyclic electron transfer [12,13] suggested that two types of cytochrome c2, soluble and membrane- bound, were present in this system. However, identification of the membrane-bound cytochrome c 2 as cytochrome c 1 and the well-known cytochrome b-c 2 complex as the cytochrome b-c t complex, was not made until 1980 [10]. Recent isolation of the cytochrome b.c 1 complex preparations [14-16] from Rhodobacter sphaeroides (formerly

0005-2728/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

204

called Rhodopseudomonas sphaeroides ) further confirmed the role of cytochrome c I in this region of the photoelectron-transfer chain.

Cytochrome c 1 of Rb. sphaeroides was identi- fied as a protein of molecular weight 30000 by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of lysed chromatophores [10] and of the purified cytochrome b-cl complex [14,16]. The mid-point redox potential was estimated to be 290 mV [10]. It is similar to that of mammalian cytochrome c 1.

In order to understand the reaction mechanism of photosynthetic electron transfer and energy conservation, it is necessary to elucidate the molecular structure and function of the participat- ing components. Recently, we developed a simple method [17] to isolate an active, pure cytochrome c~ from purified cytochrome b-c 1 complex of Rb. sphaeroides -26. In this paper we report properties of this purified bacterial cytochrome cp and com- pare them, where appropriate, with those of mitochondrial cytochrome c v

Materials and Methods

The cell culture of Rb. sphaeroides R-26 was a gift from Drs. Okamura and Feher, Department of Physics, University of California at San Diego. The growth conditions were exactly as reported [18]. The cytochrome b-c 1 complex was prepared and assayed as reported previously [16]. Calcium phosphate was prepared according to Jennerr [19]. This preparation was aged for 1 month in the cold room before it was mixed with an equal weight of cellulose powder and used for column chromatography. Horse cytochrome c, type III, and p-chlo- romercuriphenylsulfonic acid (PCMPS) were products of Sigma, Triton X-100 was from Rohm and Haas Co. Other chemicals were of the highest purity commercially available.

Absorption spectra were measured in a Cary spectrophotometer, model 219. Circular-dichroism spectra were measured in a Jasco spectropolarime- ter, model J-500. Ellipticities are expressed in de- grees per cm 2 per decimol protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis [20] and determination of cytochrome [3] and protein concentration [21] were performed by the reported methods. Sulfhydryl group determination was es-

sentially according to Boyer [22], except that PCMPS was used. The sulfhydryl content was assayed by determining the increase in absorbance at 240 nm in 50 mM phosphate buffer (pH 7.4), using a millimolar extinction coefficient of 10.3, obtained with cysteine as a standard. Fluorescence spectra were determined with a Perkin-Elmer fluorescence spectrophotometer, model 650-40. Redox potent iometr ic t i trations were carried out anaerobically according to the method reported by Dutton et al. [23]. The redox mediators used were 20 /xM of N,N,N' ,N'- tetramethylphenylenedia- mine, dichlorophenolindophenol, 5-hydroxy-l,4- naphthoquinone, 1,4-naphthoquinone, phena- zinemethosulfate, ED TA and duroquinone. Sodium dithionite and potassium ferricyanide were used in reductive and oxidative titrations, respectively. The reduction of cytochrome c~ was measured by the absorption at 552 nm in a Cary spectrophotometer, model 219.

The equilibrium studies of cytochromes Q and c, and of cytochromes c I and c. Ferricytochrome solutions were prepared by addition of excess potassium ferricyanide, the excess oxidant being removed by passage through a Sephadex G-25 column equilibrated with 50 mM phosphate buffer (pH 7.4). A similar method was used for the preparation of ferrocytochrome solutions, except that sodium dithionite was used and the excess reductant was removed by passage through a Sephadex G-25 column equilibrated with oxygen- free buffer. Equilibrium experiments were carried out as described previously [24,25]. Ferrocyto- chrome c 1 and ferricytochrome c or %, or vice versa, at concentrations of around 10 /zM each, were placed in different compartments of a dividing cuvette. Two dividing cuvettes with identical contents were placed in the sample and reference holders of a Cary 219 spectrophotometer. The base line was recorded before the cytochromes in the sample cuvette were mixed. After mixing ferrocytochrome cl with ferricytochrome c or c 2, or vice versa, spectra were immediately recorded. The concentrations of cytochromes c I and c or c 2, after equilibration, were calculated by the absorption change at 554 nm (e = - 11 mM) and 548 nm (e = + 10 mM), respectively.

Photoreduetion of cytoehrome c 1. Photoreduction of cytochrome c 1 was carried out as reported

previously for mammalian cytochrome c 1 [26]. In a Thunberg cuvette was placed 0.8 ml of 6 /~M ferricytochrome c I in 50 mM phosphate buffer, pH 7.4. The cuvette was evacuated, flushed with argon five times and then suspended in the center of a Dewar flask (10 × 30 cm) with a flat window opening 7 cm in diameter. The Dewar was filled with water. A slide projector with a 500-watt lamp and an aperture of 1 cm was placed about 1 cm from the Dewar window to prevent heat accumu- lation. Temperature in the Dewar was maintained at 23 + ] ° C. The reduction was followed by peri- odically measuring the absorption spectra. Control experiments were run under the same conditions, without light.

Isolation and purification of cytochrome Q from Rb. sphaeroides R-26. Purified cytochrome c x was prepared from the cytochrome b-c I complex of Rb. sphaeroides R-26 according to the method reported previously [17] with some modification. The cytochrome b-c x complex (3.4 ml) was dialyzed against 50 mM Tris-acetate buffer (pH 7.8) overnight with one change of buffer and then precipitated with 50% ammonium sulfate saturation. Precipitates were collected by centrifugation and redissolved in 1.7 ml of 50 mM Tris-acetate buffer (pH 7.8) containing 1.5% Triton X-100 and 2 M urea. Protein concentration was approx. 5 mg/ml . The solution was incubated in ice for 20 min and frozen at - 2 0 ° for 1 h. The frozen cytochrome b-c 1 complex was thawed and applied to a calcium phosphate/cellulose column (0.8 X 4.0 cm) equilibrated with 50 mM Tris-acetate buffer (pH 7.4), containing 1.5% Triton X-100 and 2 M urea. The column was washed with 8 ml of 25 mM sodium/potass ium phosphate buffer (pH 7.4) containing 0.25% sodium cholate. Under these conditions, cytochrome b and other unwanted proteins are not absorbed by the calcium phosphate column and appear in the effluent. After washing, crude cytochrome c 1 was eluted with 0.2 M potassium phosphate buffer, pH 8.0, containing 1% sodium cholate. The cytochrome cl fractions were combined (2.1 ml) and brought to 20% ammonium sulfate saturation by adding 0.107 gm ammonium sulfate per ml solution. The mixture was stirred at 0 ° for 20 min before being centri- fuged at 40 000 x g for 20 rain to remove precipitates. The supernatant solution was brought to

205

30% ammonium sulfate saturation by adding 0.143 ml neutralized, saturated ammonium sulfate solution per ml and the precipitate formed was again removed by centrifugation. The supernatant solution, which contains cytochrome c~, was then brought to 45% ammonium sulfate saturation by adding 0.27 ml neutralized, saturated ammonium sulfate solution per ml. Cytochrome c I was recovered in the precipitate after centrifugation, dissolved in 0.2 ml of 50 mM sodium/potassium phosphate buffer (pH 7.4) and stored at - 8 0 ° until use.

Isolation and purification of cytochrome c 2 from Rb. sphaeroides R-26. The supernatant solution obtained from the KC1 and polyethylene glycol washing step during the preparation of chromatophores [16] was collected and fractionated with solid ammonium sulfate. The precipitate obtained between 50% and 100% ammonium sulfate saturation, was collected by centrifugation and dissolved in 20 mM Tris-succinate buffer (pH 8.0) to about 20 ml. This solution was then subjected to centrifugation at 40 000 X g for 30 rain. The upper layer was discarded and the lower layer was collected and subjected to ammonium sulfate fractionation. The solution was first brought to 60% ammonium sulfate saturation with neutralized, saturated ammonium sulfate solution (1.5 ml/ml) . The precipitate formed was removed by centrifugation. The supernatant solution was brought to 90% ammonium sulfate saturation with sohd ammonium sulfate (0.204 gm/ml) . The precipitate thus obtained was dissolved in 50 mM Tris-HC1 buffer (pH 8.0), containing 1 mM EDTA, and applied to a Bio Gel A-0.5 m column. This column was equilibrated and eluted with 50 mM Tris-HC1 buffer (pH 8.0), containing 1 mM EDTA. Frac- tions containing cytochrome c 2, were pooled, di- luted with an equal volume of cold water and applied to a DEAE-52 column equilibrated with 25 mM Tris-HC1 buffer (pH 8.0). This column was washed consecutively with 10 ml 25 mM Tris-HC1 (pH 8.0), containing 0, 0.1 and 0.2 M KC1. Pure cytochrome c 2 was eluted from the column at a KC1 concentration of 0.2 M. Cytochrome c 2 thus obtained was concentrated by ammonium sulfate precipitation, redissolved in 50 mM Tris-HC1 buffer (pH 8.0) and stored at - 8 0 ° C until use [381.

206

Formation of cytochrome cz-cytochrome c 2 and of cytochrome cfcytochrome c complexes. Complex formation between cytochromes c a and c 2 of Rb. sphaeroides and between cytochrome c~ of Rb. sphaeroides and cytochrome c of horse heart, was performed essentially according to the method of Chiang and King [27]. All the cytochrome preparations were dialyzed against 10 mM sod ium/ potassium phosphate buffer (pH 7.4), before the experiments were carried out. Equal molar concentrations of bacterial ferricytochromes c a and c 2, or bacterial cytochrome c I and mammalian cytochrome c, were mixed, and the volume was adjusted to 0.5 ml with 10 mM sodium/potass ium phosphate buffer (pH 7.4). The mixture was then incubated at 0°C for 1 h before it was applied to a Sephadex G-75 column equilibrated with 10 mM phosphate buffer (pH 7.4). The column was eluted with the same buffer. Fractions of 0.5 ml were collected and the absorbance at 410 nm measured. Individual cytochromes were subjected to proce- dures identical to those used in the mixed preparation to determine the elution pattern of the individual cytochromes during Sephadex G-75 column chromatography.

Results and Discussion

Purity, molecular weight, amino acid composition and stability of purified cytochrome c~

The key steps involved in isolation of cytochrome c a from cytochrome b-c a complex of Rb. sphaeroides R-26 are Triton X-100 and urea treat- ment, calcium phosphate-cellulose column chromatography, and ammonium sulfate fractionation. In order to split cytochrome c I effectively from cytochrome b by Triton X-100 and urea treat- ment, extensive dialysis of the cytochrome b-c x complex, to remove the glycerol and cholate present in the preparation, is needed. The use of the calcium phosphate/cellulose column in the purification procedure has two purposes: to separate the unwanted proteins and to replace Triton X-100 with sodium cholate and thus facilitate subsequent ammonium sulfate fractionation and ultraviolet absorption measurements. About 27% of the cytochrome cx present in the cytochrome b-ct complex was recovered in the final purified preparation (see Table I of Ref. 17).

Purified cytochrome c~ contains 30 nmol heme

c~

A!

~i l i~ i /~ i~ i~ i i i~ i~ ,~ . ¸ . . . . . . . . . . . . . . . . . . . . ~"~-~&~

Fig. 1. The SDS-polyacrylamide gel electrophoresis of purified cytochrome c I preparations from Rb. sphaeroides and bovine heart mitochondria. (A) bacterial cytochrome c~, 30 ~g; (B) mammalian cytochrome c D 30 /zg; and (C) molecular weight protein standards containing phosphorylase B (92000), bovine serum albumin (66200), ovalbumin (45000), carbonic anhydrase (31000), soybean trypsin inhibitor (21500) and lysozyme (14 400).

per mg protein and shows one protein band in the SDS-polyacrylamide gel electrophoresis (see gel A of Fig. 1). The electrophoretic mobility of isolated cytochrome c 1 is the same as that of the heme- containing polypeptide of bovine cytochrome c 1 (see gel B of Fig. 1). The functional similarity between the photosynthetic and mitochondrial electron-transfer systems, together with the fact that isolated photosynthetic cytochrome c 1 contains only one polypeptide, suggests that active mammalian cytochrome c a is indeed one polypeptide.

Purified cytochrome c I of Rb. sphaeroides is soluble in aqueous solution and exists in a penta- mer form which is similar to its mammalian counterpart [26]. The minimum molecular weight of cytochrome cl, determined by SDS-polyacrylamide gel electrophoresis, is 30 000 (see Fig. 1). The apparent molecular weight of this cytochrome in aqueous solution, in the absence of detergents, is about 150000, estimated by gel filtration column chromatography using Bio-Gel A 0.5 m.

Although isolated cytochrome c t has a molecular weight identical to mammalian cytochrome c 1, the amino acid composition of these two proteins differ significantly. Purified cytochrome c a contains 26 Asp, 17 Thr, 12 Set, 15 Pro, 31 Glu, 31 Gly, 33 Ala, 21 Val, 11 Met, 9 lie, 22 Leu, 8 Tyr, 17 Phe, 10 Lys, 5 His, 2 Try and 11 Arg all measured in mol per mol protein. The composition * given here deviates slightly from that of the preliminary report [17], but closer, although not

* For the convenience of comparison, the amino acid composition reported here has been normalized to the contents of proline and glycine obtained from the gene sequence data.

identical, to that obtained from the amino acid sequence deduced from the gene sequence study [28]. The overall polarity of this protein, expressed as the sum of mol% of the polar amino acids, is 43%, which is slightly higher than that of mammalian cytochrome c 1 (42.3%) (9.29), but lower than that of cytochrome c I from Paracoccus de- nitrificans (44.6%) [30]. Purified Rb. sphaeroides cytochrome c a has an isoelectric point of 5.7, slightly higher than that of mammalian cytochrome c] (pI = 5.4).

Isolated cytochrome c I is stable at pH between 6.5 and 9.5. A typical bell-shape pH stabilization pattern is observed. When this cytochrome is stored at pH lower than 5 or higher than 10, it becomes irreversibly denatured. A 50% denaturation was observed when cytochrome c t was stored at 0°C for 20 h at pH 5.5 or 10.5. The stability of the protein is determined by its ability to be reduced by ascorbate. No apparent denaturation occurs when this protein is stored at - 8 0 ° C for months at neutral pH.

Spectral properties Absorption spectra. The absorption spectra of

purified cytochrome c x are given in Fig. 2A. In the oxidized form, as prepared, the spectra show max- ima at 530, 409, 360 and 278 nm. Upon reduction by ascorbate, a absorption at 552.5 nm and /3 absorption at 522 nm with a shoulder at 530 nm are observed. The Soret absorption maximum of the reduced protein is at 417 nm. Carbon mono- xide does not affect the absorption spectra of cytochrome c a in either the oxidized or reduced state. Although the spectral properties of Rb. sphaeroides cytochrome c 1 are quite similar to those of cytochrome c 1 of other sources [3,5] or cytochrome f [31], no a absorption peak splitting was observed when the spectrum was taken at low temperature [14].

It was reported that mammalian cytochrome c 1 showed a broad absorption maximum at 695 nm with a millimolar extinction coefficient of about 0.8 [3]. Isolated cytochrome c a of Rb. sphaeroides also exhibits a broad band in this region (see Fig. 2B). Upon reduction by dithionite, the absorption of cytochrome cl between 600 and 750 nm decreases greatly and the broad band at 680 nm disappears.

A 417

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f OD=01

J.

I 5 5 2 . ~

~o 3~o ~o 4~o ~o ~o 66o Wavelength, nm

207

B ~~002

6~o o~o ~,o 7~o Wavelength nm

Fig. 2. Absorption spectra of purified cytochrome c 1 of Rb. sphaeroides. (A) Absorption spectra of cytochrome q in the visible and ultraviolet regions. Cytochrome q, 0.2 mg/ml in 50 mM phosphate buffer (pH 7.4), containing 0.2% sodium cholate was used. The solid ( ) and broken ( - - - - - ) lines represent oxidized and sodium ascorbate reduced forms, respectively. (B) The absorption spectra of cytochrome c 1 in the near-infrared region. Cytochrome q, 42.8/~M, in 50 mM phosphate buffer (pH 7.4) was used. ( ) Oxidized form; (-- -- --), dithionite reduced form. Spectra were measured in a Cary spectrophotometer, model 219, at 23°C.

Circular-dichroism spectra. Fig. 3A shows the circular-dichroism spectra of isolated cytochrome c t of Rb. sphaeroides. Mammalian cytochrome c't, measured under identical conditions, is included for comparison (Fig. 3B). The oxidized enzyme shows positive CD extrema at 413 and 258 nm, and the negative extrema at 350 and 283 nm.

208

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L,o:l \ . . . . . . . . . . . 2 .1on _ ,

/

4 J B 1210 ~ ~,

10104

8104

6104

4104

2104

0 -2104

-4104 2~o 3~o 3~o ~8o 4~o s~o ~o 6bo

Wovelengt h, nm

Fig. 3. Circular dichroism spectra of cytochrome c 1 of Rb. sphaeroides and bovine heart mitochondria. (A) Bacterial cytochrome ct; (B) mammalian cytochrome c 1. ( ), oxidized; (-- -- --), reduced.

Upon reduction, a split CD profile with a positive extremum at 561 nm (O = 2.0.103) and a negative extremum at 550 nm (O = 4- 103) is observed in the et absorption region. In the envelope of the 13 absorption band, at least three positive CD bands appear. The ellipticity for the main band at 525 nm is about 9. 103; this is slightly lower than that of corresponding mammalian cytochrome c v The reduced cytochrome shows a relatively simple Soret ellipticity band at 419 nm. The ellipticity for the Soret band is 8.1 • 104, which is only 60% of that of its mammalian counterpart. In the wavelength region from 270 to 400 nm, both the oxidized and reduced cytochromes exhibit negative ellipticity bands. A negative CD extremum at 350 nm is observed with reduced cytochrome, not apparent in the oxidized form, and a negative extremum at 285 nm is observed for both forms. It should be noted that mammalian cytochrome c a shows positive CD ellipticities for both forms at this (285 nm) wavelength. Two positive eUipticity bands at 263 and 258 nm are detected in the reduced cytochrome c x of Rb. sphaeroides with ellipticities of 2.9.104 and 4 .3 ,10 4, respectively; the magnitude of these bands decreases by 23% upon oxidation. The cytochrome exhibits a typical far ultra-

violet dichroism spectrum, with minima at 220 and 208 nm and a maximum at 193 nm. No significant change in the magnitude of these ellipticities is observed upon oxidation.

The similarity in absorption spectra and molecular weight between bovine and Rb. sphaeroides cytochrome c 1 preparations indicates that these proteins have similar heme environments. On the other hand, the difference in amino acid composition and circular dichroism spectra of these two cytochromes suggests that they differ in primary structure and protein conformation. Difference in protein structure between the mammalian cytochrome cl and photosynthetic bacterial cytochrome c 1 is also suggested by our recent im- munological studies showing little cross reactivity (less than 15%) between antibodies specific for Rb. sphaeroides cytochrome c 1 and antibodies specific for bovine heart cytochrome c~ (unpublished re- suits from our laboratory), as measured by inhibi- tion and ELISA assays.

Fluorescence studies of cytochrome Q. It has been demonstrated [32,33] that heme protein quenching of the fluorescence of a molecule such as 8-anilino-l-naphthalene sulfonic acid (ANS) is a measure of the exposure of the heme moiety in a given solvent. Mammalian cytochrome c~ does not quench ANS fluorescence in cholate, but causes a blue shift of 6 nm in the wavelength of maximum emission. Cytochrome c~ of Rb. sphaeroides, on the other hand, quenches ANS fluorescence in cholate, by 7% with a 3 nm blue shift of the maximum emission wavelength. Mam- malian cytochrome c and bacterial cytochrome c 2 quench ANS fluorescence in cholate by 13 and 10%, respectively. These results indicate that the degree of exposure of the heme moiety of these isolated cytochromes decreases in the following order: bovine cytochrome c 2, Rb. sphaeroides cytochrome c 2, Rb. sphaeroides cl, bovine cytochrome c 1.

Electron-transfer reaction between Rb. sphaeroides cytochromes c 1 and c 2 or Rb. sphaeroides cytochrome Q and mammalian cytochrome c. The electron transfer from Rb. sphaeroides f.errocyto- chrome c I to ferricytochrome c 2 (Eqn. 1), or to mammalian ferricytochrome c (Eqn. 2), proceeds rapidly. The equilibrium of Eqns. 1 and 2 were studied by direct spectrophotometric measure-

31C

27C

2 3 0 >

190

150

XO

X X

0

0 X X

xO X O

0 X

x 0

0 x

0 x

X X O OX

1'5 -1'0 -05 C) C~5 1'0 1'5

Log R~dd

Fig. 4. Potentiometric titration of isolated cytochrome c 1 from Rb. sphaeroides. The titration mixture contained 50 mM potassium phosphate buffer at pH 7.0, 20/~M each of dichlorophenolindophenol, N,N,N',N'-tetramethylphenylene diamine, 5- hydroxy-l,4-naphthaquinone, 1,4-naphthaquinone, duroquinone and EDTA. Potassium ferricyanide and sodium dithionite were used in oxidative (©) and reductive ( × ) titrations, respectively. The reduction of cytochrome c t was measured by absorption at 552 nm.

ment as reported for studies of electron transfer between mammal ian cytochromes c 1 and c [24,54]. The equilibrium constants for these two reactions are calculated to be 49 and 3.5, respectively.

Rb. cl 2+ + Rb. C2 3+ ~ Rb. c~ 3+ + Rb. c2 2 + (1)

Rb. Cl 2+ +bovine c 3+ ~ Rb. cl 2+ +bovine c 2+ (2)

If we use 260 mV as the midpoint potential (Em) for isolated mammalian cytochrome c [34] and 340 mV as the midpoint potential for cytochrome c 2 or Rb. sphaeroides [35], the redox potential of isolated cytochrome c~ from Rb. sphaeroides is calculated to be 228 mV, identical with that of isolated mammalian cytochrome c~ [25] and close to that reported for the Rb. sphaeroides cy tochrome b-c 1 complex [14]. Anaerobic redox potential titration of cytochrome cl in the presence of redox mediators (Fig. 4) shows a typical n = 1 titration curve with a midpoint potential of 230 mV at p H 8.0. The midpoint potential of cytochrome c I is slightly af- fected by pH. When the titration is carried out at p H 7.0, an E m of 238 mV is obtained.

Complex formation between cytochromes Q and c 2 of Rb. sphaeroides or between Rb. sphaeroides cytochrome c 1 and mammalian cytochrome c. A1-

209

though electron transfer proceeds readily between isolated cytochromes c 1 and c 2, or between Rb. sphaeroides cytochrome c 1 and mammalian cytochrome c, no stable complex is formed when both pairs of cytochromes are mixed at low ionic strength. In contrast, mammalian cytochrome cl forms a stable stoichiometric complex with mammalian cytochrome c [27] at low ionic strength (10 mM phosphate buffer, at neutral pH). The mammalian cytochrome Cl-C complex can be isolated by molecular sieve column chromatography. When isolated Rb. sphaeroides cytochrome c 1 is mixed with an equal molar concentration of either cytochrome c 2 from the same source or with mammalian cytochrome c, in 10 mM phosphate buffer (pH 7.0) and passed through a gel filtration column of Bio-gel A 0.5 m, two protein peaks, corresponding to the Rb. sphaeroides cytochromes c~ and cytochrome c 2, or to the Rb. sphaeroides cytochrome c 1 and mammalian cytochrome c, are observed. This indicates that isolated cytochrome c 1 of Rb. sphaeroides forms no stable complex with cytochrome c 2 of the same source or with mammalian cytochrome c. However, a slight de- crease (about 1 ml) in the eluting volume of cytochrome c 2 was observed when it was mixed with cytochrome c x before column chromatography, as compared to that of cytochrome c 2 alone, suggesting formation of a transient complex between these two cytochromes, tt is likely that a transient complex between cytochromes c~ and c is necessary for electron transfer. Differential scanning calorimetric studies of mammalian cytochrome c 1 [36] also suggested a transient complex formation between mammalian cytochromes c~ and c at higher ionic strength. It is likely that the lack of complex formation between isolated Rb. sphaeroides cytochromes cl and c 2 is due to the absence of certain component in the isolated protein. To test this possibility dialyzed cytochrome b-c~ complex and equal molar of cytochrome c 2 were mixed at low ionic strength and the mixture was incubated at 0 °C for 1 h before it was subjecting to a molecular sieving column chromatograph. No complex formation was detected, indicating that the lack of a stable complex formation between cytochromes c~ and c 2 is an intrinsic property of the cytochromes and is not the missing of a certain component.

210

Photoreduction of cytochrome c I . I so la ted mi tochondr ia l cy tochrome c 1 [26] as well as chloro- p las t cy tochrome f [37] undergoes slow pho to - reduc t ion in the ~ absence of exogenous reducing agent under anaerob ic condi t ions . Rb. sphaeroides cy tochrome c 1 is also pho to reduc ib l e under anaerob ic condi t ions . The rate of pho to reduc t i on is three t imes faster in Rb. sphaeroides cy tochrome c a than that of i ts mi tochondr i a l coun te rpar t . A 50% reduc t ion of cy tochrome c I requires i l lumina- t ion for 90 min at p H 7.4. U n d e r ident ica l cond i - t ions, mi tochondr i a l cy tochrome c I requires al- mos t 4 h to reach 50% reduct ion. The comple te reduc t ion of cy toch rome c a requires 5 h. W h e n air was in t roduced to the system, in the absence of l ight, the pho to r educed cy tochrome cl is s lowly oxidized. The au tox ida t ion ra te is s l ightly faster t han that of the m a m m a l i a n cy toch rome c 1.

Since no external e lec t ron d o n o r is p resen t in the system, the source of the r educ tan t for the p h o t o r e d u c t i o n of the Rb. sphaeroides cy tochrome c I mos t l ikely comes f rom the molecule itself. One a t t rac t ive cand ida t e is the su l fhydryl groups of the p ro te in moiety. I so la ted Rb. sphaeroides cyto- ch rome c 1 con ta ins 3.0 + 0.1 mol of PCMPS- t i t r a - tab le su l fhydryl g roups per mol of cy toch rome q . A d d i t i o n of P C M P S to the p r e p a r a t i o n abol ishes the pho to reduc ib i l i t y of cy toch rome ca, suggest ing tha t the SH groups of cy toch rome c 1 are e lec t ron donors for the p h o t o r e d u c t i o n of this cy tochrome. This deduc t ion is fur ther suppor t ed by the fact tha t the P C M P S - t i t r a t a b l e su l fhydryl g roups in the pho to lyzed cy toch rome cx is less than those in non -pho to lyzed sample. The a m o u n t of su l fhydry l groups decreased upon photo lys i s is p r o p o r t i o n a l to the degree of pho to reduc t i on occurred. A simi- la r observa t ion has also been m a d e in the p h o t o - reduc t ion of m a m m a l i a n cy tochrome c a [26]. De- ta i led in fo rma t ion abou t which specific su l fhydry l g roup is involved and the poss ib le phys io log ica l i m por t ance of such su l fhydryl g roup in the elec- t ron t ransfer wi th in the cy tochrome c a molecule are not yet avai lable . F u r t h e r inves t iga t ion on this subject is needed before e lec t ron t ransfer mecha- n ism can be e lucidated.

Acknowledgements

This work was s u p p o r t e d b y grants f rom U n i t e d Sta tes D e p a r t m e n t of Agr icu l tu re (82 -CRCR-1-

1049), N a t i o n a l Ins t i tu tes of Hea l th ( G M 30721) and the O k l a h o m a Agr icu l tura l Exper iment Sta- t ion (J 4811).

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characterization of purified cytochrome c1 from rhodobacter sphaeroides r-26

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