Vol. 123, No. 3, 1984

September 28, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1234-1239

PURIFICATION AND PROPERTIES OF CYTOCHROHE b FROM PHOTOSYNTHETIC BACTERIUM RItODOPSEUDOMONAS SPHAEROIDES R-26

L i n d a Yu and Chang-An Yu

Department of Biochemistry, Oklahoma State U n i v e r s i t y Stillwater, Oklahoma 74078

Received August 24, 1984

SUMMARY-- Cytochrome b of R_~. sphaeroides R-26 has been purified from the isolated cytochrome _b-c_1 complex to homogeneity. The purification procedure involves Triton X-IO0 and urea solubilization, calcium phosphate column chromatography at different pH values, and ammonium sulfate fractionation. The purified protein contains 23 nmol heme per mg protein and has an apparent molecular weight of 43,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresls. The spectral characteristics of purified cytochrome b are similar to those of cytochrome b in the active cytochrome b-_c I complex but with a lower absorbance. The amino acid composition has been determined and compared with cytochrome b purified from other sources. © 1984 Acad~ic Press, Inc.

Cvtochrome b, an essential component of photosynthetic and respiratory

electron transport systems, has been the subject of intensitive investigation

for several decades (1,2). Attempts to isolate cytochrome b from various I

sources have, so far, resulted in somewhat modified preparations (3-8). The

spectral blue shift, decreased redox potential, and reactivity with carbon

monoxide have generally been observed once cytochrome b is split from the active

cytochrome _b-c_1 complex. While the molecular properties of cytochrome _b are

still obscure, the amino acid sequence of apocytochrome b from many species has

been determined by gene sequencing (9,10). The molecular weight of

apocytochrome b proteins, determined by the gene sequences, is around 42,000

(11), a value much higher than any obtained for the isolated cytochrome

proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Recently we have prepared pure cytochrome b, with a molecular weight of

43,000, from the cytochrume _b-c_l complex of ~R" sphaeroides R-26. It contains

one mole heme per mole protein and shows partial reconstitutive activity with

the cytochrome b deficient cytochrome b-c I complex.

In this communication we report the detailed isolation procedure for and

some properties of cytochrome b from R_~. sphaeroides R-26.

0006-291X/84 $1.50 Copyright © 1984 by Academic Press, Inc. Allrightsofreproduction in anyform reserved. 1234

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MATERIALS AND METHODS

The cell culture of R_c _. sphaeroides R-26 was a gift from Drs. Okamura and Feher, Dept. of Physics 9 UCSD. The growth conditions were exactly as reported (12). The preparation of chromatophores and the cytochrome ~-~1 complex was carried out according to methods developed in our laboratory (13). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (14), determination of cytochrome content (15) and protein concentration (16) were performed by the reported methods. Calcium phosphate was prepared according to Jenner (17). Protein molecular weight standards, containing phosphorylase B (92,000) 9 bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean Trypsin inhibitor (21,500) and cytochrome c (12,400), were purchased from BIO-RAD. Spectral measurements were done Tn a Cary spectrophotometer, model 219.

RESULTS AND DISCUSSION

Purification of C~tochrome b--- The cytochrome ~-~i complex of R__ t.

sphaeroides R-26 is dialyzed against 50 mM Tris-Acetate buffer, pH 7.8,

overnight, with one change of buffer, and brought to 50% saturation with

ammonium sulfate. The precipitates are collected by centrifugation and

re-dissolved in 50 mM Tris-acetate buffer, pH 7.8, containing 1.5% Triton X-100

and 2 M urea (TUT), to a protein concentration of approximately 5 mg/ml. This

solution is incubated at 0 °C for 20 min and then frozen at - 20 °C for 2 hrs.

The frozen solution is thawed and applied to a column (0.8 x 4.0 cm) packed with

a mixture of calcium phosphate and cellulose (i : i), and equilibrated with TUT

buffer. The ratio of sample volume to column bed volume is about i. Cytochrume

is in the effluent; and cytochrome ~I is retained on the column. Then

cytochrome ~I is eluted with 0.2 M phosphate buffer, pH 8.0~ and saved for

further purification (18). The first effluent is immediately adjusted to pH 6.5

with 2 N acetic acid, and applied to another calcium phosphate : cellulose

column, equilibrated with 50 mM Tris-acetate buffer, pH 6.59 containing 0.2%

Triton X-100. The effluent, containing crude cytochrome b, is adjusted to pH

8.0 with 2 N Tris-Base and subjected to ammonium sulfate fractionation. The

solution is brought to 25% ammonium sulfate saturation (0.133 gm/ml) and stirred

at 0 °C for 20 min before centrifugation at 40,000 x ~ for 20 min to remove the

precipitate. The supernatant solution obtained is brought to 36.5% ammonium

sulfate saturation (0.065 gm/ml) and the precipitate formed again removed by

centrifugation. This supernatant solution is then brought to 56.5% ammonium

sulfate saturation (0.14 gm/ml) and cytochrome b is recovered in the precipitate

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Table I. Summary of Purification Data

Treatment Volume Protein Cytochrome b Conc. Recovery

ml mg/ml ~ %

Cytochrome ~-~I complex I0.0 4.7 37 I00

Ist Calcium Phosphate Effluent 11.3 3.1 30 92

2rid CalciumPhosphate Effluent 13.8 1.9 22 82

AmSO4 Fractionation , 37-57% sat'd 1.0 6.0 138 37

after centrifugation. This cytochrome b is dissolved in 20 mM Tris-succinate,

pH 8.0, containing i mM EDTA and is stored at - 70 °C until use. A summary of

purification data is given in Table I. About 37% of the cytochrome b present in

the cytochrome ~-~1 complex is recovered in the final purified preparation.

Properties of Purified Cytochrome b-- Purified cytochrome ~9 as prepared,

is soluble in aqueous solution without detergent. A trace amount of Triton

X-100 is still bound to the protein, as revealed by absorption in the UV region

(see Fig.2). Purified cytochrome b contains 23 nmol heme per mg protein and

shows only one protein band in the $DS-PAGE (see Fig. I). The electrophoretic

Fig. I. The SDS-PAGE of purified cytochrome b. Gel (A), 60 ng of cytochrome b; gel ~-~-~ 20 ~g of cytochrome b and molecular Weight protein standards; gel (C)~ molecular weight protein standards.

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O.D:O.I 1

~ / ~ 'L/ ~ L ^

250 350 450 550 650 Wavelength, nm

Fil~. 2. Absorption spectra of pu r i f i ed cytochrome b of R. sphaeroides R-26. Purified cytochrome b, 0.2 mg/ml, in 20 mN Tris-Succinate, pH 8.0p containing 0.4 M ammonium sulfate was used. The solid (--) and broken (- -) lines represent oxidized and sodium dithionite reduced forms, respectively.

mobility of purified cytochrome b, relative to cytochrome ~, is 0.43, which is

identical to that of the slowest moving band in isolated cytochrome ~-~I complex

(13). The molecular weight of isolated cytochrome b, determined by SDS-PAGE,

is 43,000, which is slightly higher than that estimated for partially purified

cytochrome b from R_~. sphaeroides GA (19) but lower than our previous estimate

(13). Cytochrome b, as prepared, is in an aggregated form. The apparent

molecular weight in aqueous solution, in the absence of detergent, is over

120,000, estimated by gel filtration column chromatography using Bio-Gel A 0.5

M. The purified cytochrome b partially restores the ubiquinol-cytochrome

reductase activity to the cytochrome b-deficlent cytochrome ~-~i complex.

Fig. 2 shows the spectral properties of purified cytochrome b. The

oxidized form shows a Sorer absorption at 414 nmwith less defined spectral

characteristics at the a- and B- regions. Upon reduction by dithionite, the

absorption at 561 nm and ~absorption at 530 nm are observed. The Sorer

absorption maximum of the reduced protein is at 428 rim, which is about a 2 nm

blue shift compared to that of cytochrome _b in the active cytohrome _b-c_1

complex. Although no change in the absorption maxima of the ~-and B-regions is

observed with isolated cytochrome b, the absorbance in the a9 ~regions is

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Vol. 123, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table II. Comparison of Amino Acids Composition of Cytochrome b Purified from Various Sources

Amino Acid Mole Percent*

R.~phaeroldes Beef (3) Beef (4) Yeast (6) N. crassa (8)

A s p a r t i c Acid 4 .6 7.9 7.5 7.9 8.3

Threoa ine 4 .9 6 .4 7.6 2 .8 4 .7

Se r ine 4 .6 5 .8 5.9 5.3 8.3

Proline 10.3 6.0 5.9 5.0 4.9

Glutamlc Acid 11.6 6.6 3.5 4.0 6.1

Glycine 7.5 7.9 6.6 7.9 7.3

Alanine 12.9 7 .9 7.2 6 .5 7.5

Half-Cystlne - 2.4 0.9 0 0.8

Valine 6.3 5.1 4.7 9.6 6.7

Methionine 2.9 3.3 3.8 3.9 2.3

Isoleucine 3.7 7.6 9.4 9.4 8.6

Leucine 5.0 13.3 15.7 I I . I 12.4

Tyrosine 1.3 3.6 4.0 5.4 4.1

Phenylanine 4.7 5.5 6.2 8.8 6.4

Lys ine 7 .8 3.7 2.7 3.1 2.9

Histidine 1.7 3.0 3.1 3.4 2.4

Arginlne 5.6 2.9 2.2 4.0 3.6

Tryptophan - 2.6 3.0 1.8 2.7

*Assuming 95% amino acid recovery without halfcystine and tyrptophan.

decreased about 20% as compared to that of cytochrome b in the active cytochrome

~-~i complex. The millimolar extinction coefficient, eSm~60-570 nm of reduced

minus oxidized form is calculated to be 22.7 based on the heme content

determined by the pyridine hemochromogen spectra (15).

Table II compares the amino acid composition of purified cytochrome b from

various sources. Although the molecular weight of cytochrome ~ from R:_.

sphaeroides R-26 is very close to the value deduced from the nucleotide sequence

of apocytochrome b proteins of various species (iI)~ the amino acid compostion

of this purified cytohrome b bears little resemblance to those calculated from

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neucleotide sequence or those determined for the isolated cytochrome b of beef

(3, 4) , yeas t (67 10) and Neurospora (8) .

ACKNOWLEDGEMENT S

We are grateful to Dr. T-H Liao and Mr. H. Paudel for amino acid analysis. This work is supported partly by grants from NIH (30721), USDA (82-CRCR-I-1049) and Oklahoma Agricultural Experiment Station.

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