646
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
NO is being reduced a transient heme-NO complex is formed. 38 The cytochrome component has not yet been purified or isolated in quantity sufficient for characterization. Nitrous Oxide Reduction
Although evidence has been presented for participation of c-type cytochrome(s) in the reduction of N20, there has been no separation or purification of components of the complex NzO reductase system, a9 It is known, however, that acetylene selectively blocks the functioning of N20 reductase. 4° N2 is the product of N20 reductase in all denitrifiers capable of this activity. Acknowledgment Supported in part by National Science Foundation grant No. DES 74-21338. 38 C. D. Cox, Jr., W. J. Payne, and D. V. DerVartanian, Biochim. Biophys. Acta 253, 290 (1971). 39 T. Matsubara, J. Biochem. (Tokyo) 77, 627 (1975). 40 W. L. Balderston, B. Sherr, and W. J. Payne, Appl. Environ. Microbiol. 31,504 (1976).
[61] P u r i f i c a t i o n o f E l e c t r o n - T r a n s f e r Pseudomonas t
Components
from
B y D A V I D C . WHARTON
A number of oxidation-reduction components can be isolated from cells of Pseudomonas aeruginosa. Among the earliest to be purified and studied is the so-called Pseudomonas cytochrome oxidase, which was shown by Okunuki and his associates z-6 to catalyze the oxidation of two other cellular constituents, namely, a c-type cytochrome (cytochrome c551) and a copper-containing protein of the blue type (azurin). The rei Supported by National Institutes of Health Research Grant HL10633. 2 T. Horio, J. Biochem. (Tokyo) 45, !95 (1958). 3 T. Horio, T. Higashi, H. Matsubara, K. Kusai, M. Nakai, and K. Okunuki, Biochim. Biophys. Acta 29, 297 (1958). 4 T. Horio, T. Higashi, T. Yamanaka, H. Matsubara, and K. Okunuki, J. Biol. Chem. 236, 944 (1961). 5 T. Yamanaka, S. Kijimoto, K. Okunuki, and K. Kusai, Nature'(London) 194, 759 (1962). 6 T. Yamanaka and K. Okunuki, Biochim. Biophys. Acta 67, 379 (1963).
[61]
P U R I F I C A T I O N OF E L E C T R O N - T R A N S F E R C O M P O N E N T S
647
duced form ofPseudomonas cytochrome oxidase in turn can be oxidized either by molecular oxygen or by nitrite. Since Pseudomonas cytochrome oxidase is synthesized by the organism only anaerobically in the presence of nitrate or nitrite it was proposed that the enzyme in vivo is really a nitrite reductase. 6 Nonetheless, the term Pseudomonas cytochrome oxidase has been retained. In addition to the oxidase, cytochrome c-551, and azurin, an additional c-type cytochrome, called cytochrome c-556, can be purified from extracts of P. aeruginosa, r Pseudomonas Cytochrome
Oxidase
Ferrocytochrome c-551 + 2H ÷ + 1/202 ~ ferricytochrome c-551 + H20 or
Cuprous azurin + 2H + + 1/202 ~ cupric azurin + H20
Pseudomonas cytochrome oxidase has been isolated in a homogeneous crystalline form from Pseudomonas aeruginosa by Okunuki and his co-workers, 2-4 by Kuronen and Ellfolk, s and by Gudat, Singh, and Wharton. 7 In each case the purified enzyme contains equimolar quantities of heine c and heme dl.
Assay
Method
Principle. Pseudomonas cytochrome oxidase is most conveniently assayed by the spectrophotometric method described by Gudat et al.r The rate of oxidation of Pseudomonas cytochrome c-551 is measured by following the decrease in the absorbance of its a-band at 551 nm. Alternatively, the rate of oxidation of reduced azurin can be measured by observing the increase in its absorbance at 625 nm. Reagents 1. Potassium phosphate buffer, 0.1 M , p H 6.0. 2. Pseudomonas ferrocytochrome c-551: Cytochrome c-551 is purified from P. aeruginosa and is maintained as a stock solution of 200/xM in 0.1 M potassium phosphate buffer, pH 6.0. A few milliliters of the stock solution are reduced by adding a few grains of sodium dithionite; excess dithionite is removed by gently shaking the solution for a few minutes. The solution should be used only on the day it is reduced. r j. C. Gudat, J. Singh, and D. C. Wharton, Biochim. Biophys. Acta 292, 376 (1973). T. Kuronen and N. Ellfolk, Biochim. Biophys. Acta 275, 308 (1972).
648
OTHER MICROBIALELECTRONTRANSPORTSYSTEMS
[61]
3. P s e u d o m o n a s azurin (reduced): Azurin is purified from P. aeruginosa and is maintained as a stock solution of 500 t~M in 0.1 M p o t a s s i u m p h o s p h a t e buffer, p H 6.0. A few milliliters of this stock solution can be reduced and handled exactly as in the case of P s e u d o m o n a s c y t o c h r o m e c-551. 4. Potassium ferricyanide, 0.1 M. 5. E n z y m e : I m m e d i a t e l y before assay, P s e u d o m o n a s c y t o c h r o m e oxidase is diluted to a protein concentration of about 5 mg/ml in 0.1 M potassium p h o s p h a t e buffer, p H 6.0 (0°-4°). Procedure. To each of two 1-ml cuvettes with a 1-cm light path add the following: 0.7 ml of p o t a s s i u m p h o s p h a t e buffer and 0.3 ml of Pseudomonas f e r r o c y t o c h r o m e c-551 or 0.3 ml of P s e u d o m o n a s azurin. The blank cuvette is oxidized with 0.01 ml of p o t a s s i u m ferricyanide. After t e m p e r a t u r e equilibration at 30 ° for 4 min, the reaction is initiated by adding 10/~1 of e n z y m e solution. F o r the oxidation of f e r r o c y t o c h r o m e c-551 the decrease in a b s o r b a n c e is m e a s u r e d at 551 nm; for the oxidation of azurin the increase in a b s o r b a n c e at 625 nm is measured. Definition o f Activity. The activity of P s e u d o m o n a s c y t o c h r o m e oxidase m a y be defined in terms of the first-order velocity constant. Concentration(t~me o) min-' k = 2.3 log Concentration(tim~ 0 + , rain) The specific activity is calculated from the known concentration of c y t o c h r o m e c-551 or azurin and e n z y m e in the a s s a y mixture and the estimated first-order velocity constant. S.A.--
k(concentration of substrate) (concentration of enzyme)
The concentration of c y t o c h r o m e c-551 is calculated on the basis of e,~51,m = 28.3 m M -1 cm-1, 9"10 and that of azurin is based on e62,~nm = 6.95 mM-1 cm-l,10, 11 for the reduced and oxidized forms, respectively. Alternative M e t h o d s o f A s s a y . P s e u d o m o n a s c y t o c h r o m e oxidase activity can also be determined by measuring the uptake of o x y g e n in solution either polarographically or manometrically essentially as described for the assay of m a m m a l i a n c y t o c h r o m e c-oxidase.'2 9 T. Horio, T. Higashi, M. Sasagawa, K. Kusai, M. Nakai, and K. Okunuki, Biochem. J. 77, 194 (1960). 10T. Yamanaka, in "The Biochemistry of Copper" (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), p. 275. Academic Press, New York, 1966. ,1 T. Yamanaka, S. Kijimoto, and K. Okunuki, J. Biochem. (Tokyo) 53, 256 (1963). 1~D. C. Wharton and D. E. Griffiths, Arch. Biochem. Biophys. 96, 103 (1962).
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
649
Assay for Nitrite Reductase Activity. Pseudomonas cytochrome oxidase can be assayed for its nitrite reductase activity using the method of Yamanaka and Okunuki. 6 This spectrophotometric method is essentially the same as that used for measuring oxidase activity except that 0.03 ml of 0.2 M potassium nitrite is included in the assay mixture and the reaction is carried out under anaerobic conditions in Thunberg-type cuvettes. Purification Procedure Two procedures for purifying Pseudomonas cytochrome oxidase are described below. The first procedure has the advantage of conveniently producing fractions containing other oxidation-reaction components of interest that can readily be purified further to homogeneity. All operations are carried out at 0°-4 ° unless specified otherwise. Protein estimations are made by the method of Lowry et al. 13 with bovine serum albumin as the standard.
The Organism and Culture Conditions A culture of Pseudomonas aeruginosa is maintained routinely on an agar slant (2%) containing bouillon-peptone. Slants are stored in the refrigerator, and transfers made to new slants every 2 months. For the large-scale production of cells of P. aeruginosa the organism is grown in three stages after transfer from an agar slant. In the first stage 30 ml of a bouillon-peptone medium (5 g of beef extract, 10 g of peptone, and 2,5 g of NaCI per liter of solution adjusted to pH 7.0 with NaOH) are inoculated with the culture. The flask is shaken for 24 hr at 37 °, then this culture is added to 12 liters of the bouillon-peptone medium and the cells are grown under the same conditions as for the 30-ml volume. For the final stage of cell production the 12 liters of inoculum are added to 190 liters of nitrate medium (10 g of beef extract, 10 g of peptone, 20 g of KNO3, 6.4 g of KH2PO4, 3.6 g of Na2HPO4 per liter of solution) in a large fermentor, and the cells are grown at 37 ° without aeration but with slight agitation. The addition of a few milfiliters of antifoam (Union Carbide Corp., SAG471) is advisable because of frothing from the evolution of gas. When the pH of the culture reaches 8 the cells are harvested using a continuous-flow Sharpies centrifuge. After harvesting, the cell paste is collected and frozen without washing. This paste can then be stored at - 2 0 °. The yield of cells is about 2.3 g per liter of medium. As an alternative to a fermentor, cells can be grown in the nitrate 13 O. H. L o w r y , N. J. R o s e b r o u g h , A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
650
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
medium contained in autoclavable polypropylene bottles of 20-liter capacity. Harvesting of these cells can be accomplished with a refrigerated centrifuge if a continuous-flow Sharples centrifuge is not available.
Preparation of Crude Extract Four hundred grams of the frozen cell paste are thawed and suspended uniformly in 1200 ml of 0.1 M phosphate buffer, pH 6.0, containing l0 mg of deoxyribonuclease (Sigma, type I from bovine pancreas). The suspension is filtered through two layers of cheesecloth, and the cells are broken by passing the suspension through a Gaulin laboratory homogenizer (Gaulin Manufacturing Co., Everett, Massachusetts) at a pressure of 9,000 psi. As the effluent emerges from the homogenizer it is cooled to 5 ° in a cold brine bath and is rehomogenized once. The cooled effluent of the second homogenization is centrifuged for 40 rain at 18,000 g. The brownish supernatant is decanted and stored at 4 °, and the residue is resuspended in 600 ml of the phosphate buffer. This suspension then is rehomogenized in the Gaulin homogenizer. The cooled effluent is centrifuged for 40 rain at 18,000 g, and the supernatant is combined with that of the first centrifugation; the residue is discarded. As an alternative to the Gaulin homogenization an extract may be prepared from an acetone-dried powder of the cells essentially as described by Kuronen and Ellfolk s or by Horio. 2 In this procedure 100 g of acetone-dried cells are stirred in 950 ml of 0.1 M phosphate buffer, pH 7.0, containing 2 mg of deoxyribonuclease, for 3 hr at 5 °. This suspension then is centrifuged for 40 min at 18,000 g and the supernatant is recovered.
Purification Procedure 7 Step 1. Fractionation with Ammonium Sulfate. The combined extract is brought to 40% of saturation by adding solid (NH4)2SO4 (29.0 g/100 ml). This solution is centrifuged for 30 min at 18,000 g. The golden-brown supernatant is decanted and is brought to 95% of saturation by adding solid (NH4)2SO4 (39.8 g/100 ml). This solution is centrifuged for 20 rain at 18,000 g, and the supernatant is discarded. The light tan residue is suspended in 150 ml of distilled HzO and dialyzed (Visking No. 30 dialysis tubing) for 12 hr with two changes against 6 liters of 10 mM phosphate buffer, pH 6.0. Step 2. Sephadex G-IO0 Chromatography. The dialyzed solution is applied to the base of a column (5.0 cm × 100.0 cm) of Sephadex G-100 that is equilibrated with 10 mM phosphate buffer, pH 6.0. The column is
[61]
651
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
eluted with the same buffer by upward flow, and 6-ml fractions are collected by means of a fraction collector. A typical elution pattern, illustrated in Fig. 1, shows the separation of the Pseudomonas cytochrome oxidase from the other oxidation-reduction components contained in the extract. These fractions are retained for further purification.
Step 3. CM-Sephadex C-50 Chromatography. Fractions from the column of Sephadex G-100 that contain cytochrome oxidase activity are combined and applied to the top of a column (2.5 cm × 40.0 cm) of CMSephadex C-50 that is equilibrated with 10 mM phosphate buffer, pH 6.0. The bulk of the greenish-brown Pseudomonas cytochrome oxidase does not adhere to the CM-Sephadex and is eluted with additional equilibrating buffer. Occasionally some of the oxidase may adhere to the column. If this occurs the adhering oxidase may be eluted with 0.1 M sodium phosphate buffer, pH 7.0, after first washing the column with 400 ml of 10 mM sodium phosphate buffer, pH 7.0. It is not usually necessary to use a fraction collector to obtain the oxidase that passes directly through the column. However, in the event that some of the enzyme adheres to the column it is advisable to collect the effluent of the 0.1 M phosphate buffer, pH 7.0, in 6-ml fractions. The effluent from the CM-Sephadex column with oxidase activity is combined and dialyzed (Visking No. 30 dialysis tubing) overnight with one change against 40 volumes of 50 mM Tris-chloride, pH 8.0. @
4
~
P. Cyt Oxidase
~
P. Cu Protein
~ 3
2
a
e m t~
E E
t n
P_= Cyt c SSI
c-SS6 3
II,
Is
0.3
I
2 | 9
0.4
O.S
I 0.6
I 0.7
I 0.8
0.9
1.0
Ve / Vt FIG. 1. Elution pattern of o x i d a t i o n - r e d u c t i o n c o m p o n e n t s ofPseudomonas aeruginosa from a c o l u m n of S e p h a d e x G-100. Elution was m a d e with 10 m M p h o s p h a t e buffer, pH 6.0, and s a m p l e s were collected in 6-ml fractions.
652
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
Step 4. DEAE-Cellulose Chromatography. The greenish brown dialyzate is applied to the top of a column (2.5 cm × 25.0 cm) of DEAEcellulose that is equilibrated with 50 mM Tris-chloride, pH 8.0. The Pseudomonas cytochrome oxidase adheres weakly to the DEAE-cellulose and is eluted with 50 mM Tris-chloride, pH 8.0, in 3-ml fractions. Those fractions with A410 nm : A 2 8 0 nm of greater than 1.0 are combined and dialyzed overnight with one change against 40 volumes of 20 M Trischloride, pH 8.0. Step 5. Gradient Elution Chromatography with DEAE-Celhdose. The dialyzed solution from the preceding step is applied to a column (2.0 cm × 20,0 cm) of DEAE-cellulose that is equilibrated with 20 mM Trischloride, pH 8.0. The green oxidase is eluted by means of a linear gradient of 20 mM to 50 mM Tris-chloride, pH 8.0. The total volume of the gradient buffers is 1000 ml. The effluent is collected in 3-ml fractions and those with A41onm : A 2 8 0 nm of greater than 1.15 are combined. The combined fractions are dialyzed overnight against 40 volumes of 20 mM phosphate buffer, pH 6.5, and concentrated by ultrafiltration (Amicon Diaflo PM-10 membranes or Millipore Immersible Molecular Separator). The results of a typical purification by this procedure are shown in Table I. Step 6. Crystallization. The purified Pseudomonas cytochrome oxidase can be crystallized out of (NH4hSO4. To do this the concentrated solution of enzyme from the preceding step is brought to a slight turbidity
TABLE I PURIFICATION OF Pseudomonas CYTOCHROME OXIDASE BY PROCEDURE I
Fraction Crude extract (NH4)SO4, 40-95% Sephadex G-100 CM-Sephadex DEAE-cellulose Gradient elution on DEAE-cellulose Crystallized
Volume (ml)
Total protein (mg)
Total activity (units)"
Specific activity (units/rag protein)
Purification (fold)
Yield (%)
1690 365 540 595 175 10
27,716 9,672 4,374 3,213 630 98
5,273 2,226 1,793 1,541 724 350
0.19 0.23 0.41 0.48 1.15 3.55
1.0 1.2 2.2 2.5 6. I 18.7
100 42 34 29 14 6.6
68
245
3.60
18.9
4.6
4.5
a Units are expressed as micromoles of cytochrome c-551 oxidized per minute.
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
653
by gradually adding finely powdered (NH4)2SO 4 at room temperature. The turbidity is sedimented out by centrifugation for 10 min at 18,000 g, and the supernatant is allowed to stand at room temperature. In time (124 hr) flat diamond-shaped crystals appear. The crystals can be collected by centrifugation for about 10 min in a clinical centrifuge. The sedimented crystals are dissolved in 20 mM phosphate buffer, pH 6.0.
Purification Procedure H 8 Step 1. DEAE-Celhdose Chromatography. The crude extract is dialyzed for 12 hr against 6 liters of 20 mM potassium phosphate buffer, pH 6.9. Two changes of dialysis buffer are made. The dialyzed extract is applied to the top of a column (5.0 cm × 50.0 cm) of DEAE-cellulose equilibrated with 20 mM potassium phosphate buffer, pH 6.9. The brownish red fraction containing Pseudomonas cytochrome oxidase does not bind to the DEAE-cellulose. Step 2. CM-Celhdose Chromatography. The effluent from the DEAEcellulose column is adjusted to pH 6.4 by adding a suitable amount of 20 mM K2HPO4. After dilution with an equal volume of water, this solution is applied to the top of a column (2.5 cm × 25.0 cm) of CM-cellulose equilibrated with 10 mM potassium phosphate, pH 6.4. The c-type cytochromes in the solution are not retained by the column while the oxidase binds. The column is washed with 10 mM potassium phosphate, pH 6.7, until the effluent is colorless. The Pseudomonas cytochrome oxidase is eluted with 40 mM potassium phosphate, pH 6.9. Those green fractions with oxidase activity are combined and concentrated) by ultrafiltration (Amicon Diaflo PM-10 membrane or Millipore immersible molecular separator). Step 3. Sephadex G-lO0 Chromatography. The concentrated solution is applied to the top of a column (2.5 cm × 95.0 cm) of Sephadex G-100 equilibrated with 10 mM potassium phosphate, pH 6.4. The volume of enzyme applied to the column should not exceed 10 ml. The column is eluted with the equilibrating buffer, and collection of 3-ml fractions is made. Those fractions with A410 nm "A280 nm in excess of 1.0 are combined and concentrated by ultrafiltration as in step 2. Step 4. Crystallization. The concentrated Psendomonas cytochrome oxidase may be crystallized according to the method described in Procedure I. In this case two crystallizations are recommended to achieve maximum purity. The results of a typical purification by this procedure are shown in Table II.
654
O T H E R M I C R O B I A L E L E C T R O N TRANSPORT SYSTEMS
[61]
TABLE lI PURIFICATION OF Pseudomonas CYTOCHROME OXIDASE BY PROCEDURE II" r
Fraction
Volume (ml)
Total protein (rag)
Extract DEAE-cellulose CM-cellulose Sephadex G-100 Crystallized Recrystallized
950 1076 7.6 42 0.8 0.5
5900 1830 66 21 10.9 7.8
Total activity (units)t' 247 162 93 53 35 30
Specific activity (units/mg Purification Yield protein) (fold) (%) 0.04 0.09 1.40 2.52 3.22 3.78
1.0 2.1 33 66 77 90
100 66 38 21 14 12
" Data are from T. Kuronen and N. Ellfolk, Biochem. Biophys. Acta 275, 308 (1972). b Units are expressed as micromoles of cytochrome c-551 oxidized per minute. Properties
Storage and Stability. The purified Pseudomonas c y t o c h r o m e oxidase m a y be stored frozen at - 2 0 ° or below for several w e e k s without significant loss of oxidase or nitrite reductase activity. H o w e v e r , r e p e a t e d freezing and thawing of a sample should be avoided since this results in a noticeable loss of these activities. Purity. The Pseudomonas c y t o c h r o m e oxidase p r e p a r e d by either p r o c e d u r e is h o m o g e n e o u s according to p o l y a c r y l a m i d e gel electrophoresis as well as on the basis of data obtained from sedimentation equilibrium analysis. 7 Composition and Spectral Properties. The absorption spectra of Pseudomonas c y t o c h r o m e oxidase in its oxidized and reduced f o r m s are shown in Fig. 2. In the reduced form the absorption m a x i m a at 549-554 nm, 521 nm, and 418 nm represent the ~-, fl-, and Soret-bands, respectively, of the h e m e c constituent, and those at 620-660 nm and 460 nm represent the a- and Soret-bands, respectively, of the second heine group, h e m e di. T h e s e two heme groups are present in a p p r o x i m a t e l y equimolar amounts and account for o v e r 90% of the metal found in the e n z y m e . The millimolar extinction coefficients of the various m a x i m a are given in Table III. The E P R s p e c t r u m of Pseudomonas c y t o c h r o m e oxidase is shown in Fig. 3. The signals with g values at 2.93, 2.31, and 1.4 can be attributed to the ferric iron of the heine c constituent, and those at 2.45 and 1.71 can be associated with the ferric iron of the h e m e dl in the e n z y m e .
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
!
I
655
I
0.8
0.6 ® u o .-nO4 o
0.2
0
t /
,/
I 400
,\
I 500
Wavelength(nm)
I 600
FIG. 2. Absorption spectra of P s e u d o m o n a s cytochrome oxidase. The e n z y m e was dissolved in 0.1 M p h o s p h a t e buffer, pH 6.5. - - -, T h e oxidized portion; - - , the preparation reduced with Na~2S204.
Those signals with g values at 4.40 and 2.08 are due to contaminating iron and copper, respectively. The ferrous form of the oxidase is diamagnetic and has no EPR spectrum.
Molecular Weight. Pseudomonas cytochrome oxidase has a molecular weight of about 120,000. 7' s The minimal molecular weight on the basis of heme iron content is approximately 34,000. This result suggests the presence of 4 heme groups per 120,000 Mr, or 2 heme c and 2 heme dl groups. T A B L E llI ABSORPTION M A X I M A AND E X T I N C T I O N C O E F F I C I E N T S OF R E D U C E D
Psettdomonas
CYTOCHROME OXIDASE a
Heme group Heme c
H e m e d~
Wavelength (nm)
et' (raM -~ cm -~)
544 549 521 418 655 620 460
29.7 30.2 27.7 182 24.7 24.0 56.0
" Data from T. Horio, T. Higashi, T. Y a m a n a k a , H. Matsubara, and K. Okunuki, J. Biol. C h e m . 236, 944 (1961) and from D. C. W h a r t o n (unpublished).
b Extinction coefficients of the h e m e d~ moiety are very sensitive to pH; those given are for p H 6.5 in 0.I M phosphate.
656
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
l
[61]
gi2.45
H
g=2.931t
gi1.4 ~
Amplification
FIG. 3. Electron paramagnetic resonance (EPR) spectrum of oxidized Pse,domonas cytochrome oxidase. The instrument settings were: power output, 0.3 roW; field modulation, 5 G; temperature, 13°K; microwave frequency, approximately 9250 MHz.
The native oxidase can be dissociated by sodium dodecyl sulfate into two subunits each of about Mr 60,000. The finding '4 that only one Nterminal amino acid, a lysine residue, is present in the protein and that there are only two cysteine residues per Mr 60,000 suggests that the two subunits may be identical in primary structure. Thus, each subunit may contain one heme c group and one heme dl group. Oxidation-Reduction Potentials. The midpoint potentials of the heme c and heme dl constituents of Pseudomonas cytochrome oxidase are +0.288 V and +0.218 V, respectively. '5' ,6 Reaction with Pseudomonas Cytochrome c-551 and Azurin. Pseudomonas cytochrome oxidase accepts electrons from either Pseudornonas cytochrome c-551 or azurin. Maximum activity has been reported at pH 6.0 by Gudat et al. 7 and at pH 5.3 by Kuronen and Ellfolk. 8 With oxygen as the final electron acceptor, the K m for Pseudomonas cytochrome c551 is 10.5 /zM at pH 6.0 and 30 ° while the Km for azurin is 17.2 /xM under the same conditions. Reaction with Molecular Oxygen and Nitrite. Reduced Pseudomonas 14 y. Nagata, T. Yamanaka, and K. Okunuki, Biochim. Biophys. Acta 221, 668 (1970). ,s T. Horio, M. D. Kamen, and H. de Klerk, J. Biol. Chem. 236, 2783 (1961). 16 D. C. Wharton, J. C. Gudat, and Q. H. Gibson, Biochim. Biophys. Acta 292, 611 (1973).
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PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
657
cytochrome oxidase donates electrons either to molecular oxygen or nitrite. Turnover numbers of 107 mol and 139 mol of cytochrome c-551 oxidized rain -1 tool enzyme -1 with O~ and with nitrite, respectively, have been reported by Kuronen and Ellfolk s at 23 ° and a pH of 6.5. Circumstantial evidence has been presented 17 to indicate that the product of the reduction of 02 in these reactions is H20. The product of the reduction of nitrite is NO on the basis of spectral evidence, is During in vitro reduction of nitrite the enzyme becomes gradually inhibited, with the concomitant formation of a spectral form indistinguishable from that created by adding NO to a sample of the reduced oxidase. Inhibitors. Both cytochrome oxidase and nitrite reductase activities are inhibited 100% by 0.1 mM cyanide. NO and N~- also inhibit these activities. Carbon monoxide (CO--O2, 9: 1) causes a 90% inhibition of oxidase activity but has no inhibitory effect, even at 100% CO, on nitrite reductase activity. Nitrite is a competitive inhibitor of oxygen consumption. 3 Pseudomonas C yt ochr om e c-551 and Azurin F e r r i c y t o c h r o m e c-551 + e
--~ f e r r o c y t o c h r o m e c-551
C u p r i c a z u r i n + e - -~ c u p r o u s a z u r i n
Purification P r o c e d u r e For the purification of Pseudomonas cytochrome c-551 it is convenient to begin with the fractions that elute as a broad band (VeNt = 0.750.95) from the column of Sephadex G-100 (see step 2 of the purification procedure I for Pseudomonas cytochrome oxidase). These fractions contain both Pseudomonas cytochrome c-551 and azurin. Step I. Acid Precipitation. The combined fractions from the Sephadex G-100 column are adjusted to a pH of 3.9 by stirring in a suitable volume of 50 mM acetic acid. The resulting turbid solution is centrifuged for 20 rain at 20,000 g and the clear, grayish supernatant is decanted; the whitish precipitate is discarded. Step 2. CM-Cellulose Chromatography. The clear supernatant from step 1 is mixed with a few milliliters of 0.1 M potassium ferricyanide and is applied to the top of a column (1.5 cm × 25.0 cm) of CM-cellulose that is equilibrated with 50 mM acetate-NH4OH buffer, pH 3.9. The column J~T. Yamanaka, A. Ota, and K. Okunuki, J. Biochem. (Tokyo) 49, 414 (1961). is T. Yamanaka and K. Okunuki, Biochim. Biophys. Acta 67, 394 (1963).
658
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
is washed with about 400 ml of the equilibrating buffer, the Pseudomonas cytochrome c-551 is eluted as a red band with 50 mM acetate-NH4OH buffer, pH 4.45. After complete elution of the Pseudomonas cytochrome c-551, the column is washed with an additional 100 ml of the 50 mM acetate buffer, pH 4.45. The blue azurin, which by now has migrated about half-way down the column, is eluted next with 50 mM acetateNH4OH buffer, pH 4.65. At this stage each major component is contaminated by a small amount of the other. Almost all of this contamination can be removed by rechromatography on CM-cellulose.
Step 3. Rechromatography on CM-Cellulose. The solutions of Pseudomonas cytochrome c-551 and of azurin are each adjusted to a pH of 3.9 by adding a suitable volume of 50 mM acetic acid. Each solution is then added to the top of separate columns (1.5 cm x 25.0 cm) of CMcellulose equilibrated with 50 mM acetate-NH4OH buffer, pH 3.9. The Pseudomonas cytochrome c-551 in the one column is eluted using 50 mM acetate-NHaOH buffer, pH 4.40. The red effluent is dialyzed with two changes for 12 hr against 40 volumes of 0.1 M phosphate, pH 6.0, and concentrated by membrane ultrafiltration (Amicon Diaflo PM-10 or Millipore immersible molecular separator). The second column of CM-cellulose, adsorbing the blue azurin, first is washed with 400 ml of 50 mM acetate-NH4OH buffer, pH 4.45. The azurin is eluted as a blue band using 50 mM acetate-NH4OH buffer, pH 4.65. The blue effluent is dialyzed with two changes for 12 hr against 40 volumes of 0.1 M phosphate buffer, pH 6.0, and concentrated by membrane ultrafiltration as in the case of the cytochrome c-551. Step 4. (a) Crystallization of Pseudomonas Cytochrome c-551. The solution of Pseudomonas cytochrome c-551, concentrated in the preceding step, is first assayed for its absorbance at 551 nm using a suitably diluted aliquot reduced with a few grains of sodium dithionite. The A551nm c m - 1 of the undiluted solution must be at least 30 before crystallization is attempted. If the concentration is of sufficient strength, a fine powder of (NH4)2SO4 is added in small portions at room temperature until a slight turbidity appears. The pH of the solution is maintained at about 7.5 by the addition of dilute NH4OH. The turbid solution is centrifuged for 10 rain at 10,000 g at room temperature, and the clear red supernatant is decanted. This supernatant is reduced by adding a few grains of sodium dithionite. This solution becomes turbid and upon standing overnight at room temperature is centrifuged for 10 min in a clinical centrifuge at room temperature to collect the crystals. The supernatant, which contains about 30% of the original Pseudomonas cytochrome c-551, is decanted and should be retained. The fine needle-shaped crystals can be dissolved
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
659
in a small amount of cold H20 and can be rid of residual (NH4)2804 by passing the solution through a small column (1.0 c m x 15.0 cm) of Sephadex G-25 equilibrated with 0.1 M phosphate buffer, pH 6.0.
(b) Crystallization of Pseudomonas Azurin. The absorbance at 625 nm of the azurin obtained in step 3 is determined using a suitably diluted aliquot of the oxidized preparation. If the A625nm of the undiluted solution is at least 10, then the crystallization procedure can be commenced. To do this a fine powder of (NH4)2SO4 is added gradually until a slight turbidity is observed. The pH is maintained at about 7.0 during this period by adding dilute NH4OH from time to time. After 2 hr the slightly turbid solution is centrifuged for 10 min at 10,000 g at room temperature, and the clear blue supernatant is decanted. A small amount of finely powdered (NH4)zSO4 is added to the supernatant solution until a slight turbidity begins to form. This solution is allowed to sit at room temperature for a day or two, during which time dark blue, needle-shaped crystals form. The crystals are harvested by centrifugation for 10 min in a clinical centrifuge at room temperature. The blue supernatant can be retained. The crystalline pellet is dissolved in a small volume of cold H20 and separated from residual (NH4)2SO4 by chromatography on a small column (1.0 x 15.0 cm) of Sephadex G-25 equilibrated with 0.1 M phosphate buffer, pH 6.0.
Properties of Pseudomonas Cytochrome c-551 and Azurin
Storage and Stability. Buffered solutions of Pseudomonas cytochrome c-551 and azurin can be stored at - 2 0 ° or below for months without noticeable deterioration. Lyophilized samples kept for up to 2 years at - 2 0 ° or below have retained their normal reactivities with Pseudomonas cytochrome oxidase and exhibit no alteration of their spectral properties. Purity. Samples of both Pseudomonas cytochrome c-551 and azurin that have undergone two chromatographic steps on CM-cellulose followed by crystallization exhibit only a single protein-staining band following polyacrylamide gel electrophoresis. Spectral Properties. The absorption spectra of Pseudomonas cytochrome c-551 in its ferric and ferrous forms are shown in Fig. 4. The ferrous form is characterized by absorption maxima in the visible region at 551,521, and 416 nm. On the basis of the iron content E551n m has been calculated to be 28.3 mM -1 cm -1 for the reduced form. 9"lo The absorption spectra of Pseudomonas azurin in its cupric and cuprous forms are
660
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS 0.75
t
[61]
u
=~o.so i,~_ ,< 0.25
0
I I 400 500 Wavelength (nm)
600
FIo. 4. Absorption spectra of Pseudomonas cytochrome c-551. The protein was dissolved in 0.1 M phosphate buffer, pH 6.5. - - -, The oxidized portion; , the preparation reduced with Na25204.
illustrated in Fig. 5. The cupric form shows a broad absorption maximum near 625 nm and a very broad and very small absorption between 325 nm and 450 nm. The reduced or cuprous azurin has no absorption maxima in the visible region. On the basis of the copper content %25nm has been calculated to be 6.95 mM -1 cm -1 for the cupric protein. 10, 11
Molecular Weight. On the basis of its iron content, the minimal molecular weight of Pseudomonas cytochrome c-551 has been calculated to be 8100, and a value of 7600 has been obtained from sedimentation velocity data. 9' 10 These results indicate the presence of one heme group per protein molecule. The molecular weight of Pseudomonas azurin has been determined
I
I
I
/f ~',\\
0.6
iI
i0.4 .-0
\',
/ /
~=)
ii
~[ 0.2
\\
\\ \x
/" i//
~00 I
x~
600 I
Wavelength
760
(nm)
~do
F I G . 5 . Absorption spectra ofPseltdornonas azurin. The protein was dissolved in 0 . 1 M phosphate buffer, pH 6 . 5 . - - -, The oxidized portion; - - , the preparation reduced with
Na~25204.
[61]
PURIFICATION OF ELECTRON-TRANSFERCOMPONENTS
661
to be about 17,400 from sedimentation velocity data and about 16,600 on the basis of copper content.l°' 11 These data support the conclusion that each protein molecule contains one atom of copper.
Oxidation-Redaction Potentials. The oxidation-reduction midpoint potentials of Pseudomonas cytochrome c-551 and azurin at pH 6.4 have been measured as +0.286 V and +0.328 V, respectively. 1° Electron-Transfer Reactions. Both reduced Pseudomonas cytochrome c-551 and azurin donate electrons to Pseudornonas cytochrome oxidase. Both proteins in their oxidized forms are capable of accepting electrons from NADH by means of a soluble Pseudomonas NADH dehydrogenase and certain quinones.19 In addition, the Pseudomonas cytochrome c-551 and azurin are able to transfer electrons between themselves. 2° A number of reducing agents, including N a 2 5 2 0 4 and ascorbic acid, reduce both proteins. ~9D. C. Wharton, unpublished results. ~0 E. Antonini, A. Finazzi-Agro, L. Avigliano, P. Guerrieri, G. Rotilio, and B. Mondovi, J. Bbd. Chem. 245, 4847 (1970).
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
NO is being reduced a transient heme-NO complex is formed. 38 The cytochrome component has not yet been purified or isolated in quantity sufficient for characterization. Nitrous Oxide Reduction
Although evidence has been presented for participation of c-type cytochrome(s) in the reduction of N20, there has been no separation or purification of components of the complex NzO reductase system, a9 It is known, however, that acetylene selectively blocks the functioning of N20 reductase. 4° N2 is the product of N20 reductase in all denitrifiers capable of this activity. Acknowledgment Supported in part by National Science Foundation grant No. DES 74-21338. 38 C. D. Cox, Jr., W. J. Payne, and D. V. DerVartanian, Biochim. Biophys. Acta 253, 290 (1971). 39 T. Matsubara, J. Biochem. (Tokyo) 77, 627 (1975). 40 W. L. Balderston, B. Sherr, and W. J. Payne, Appl. Environ. Microbiol. 31,504 (1976).
[61] P u r i f i c a t i o n o f E l e c t r o n - T r a n s f e r Pseudomonas t
Components
from
B y D A V I D C . WHARTON
A number of oxidation-reduction components can be isolated from cells of Pseudomonas aeruginosa. Among the earliest to be purified and studied is the so-called Pseudomonas cytochrome oxidase, which was shown by Okunuki and his associates z-6 to catalyze the oxidation of two other cellular constituents, namely, a c-type cytochrome (cytochrome c551) and a copper-containing protein of the blue type (azurin). The rei Supported by National Institutes of Health Research Grant HL10633. 2 T. Horio, J. Biochem. (Tokyo) 45, !95 (1958). 3 T. Horio, T. Higashi, H. Matsubara, K. Kusai, M. Nakai, and K. Okunuki, Biochim. Biophys. Acta 29, 297 (1958). 4 T. Horio, T. Higashi, T. Yamanaka, H. Matsubara, and K. Okunuki, J. Biol. Chem. 236, 944 (1961). 5 T. Yamanaka, S. Kijimoto, K. Okunuki, and K. Kusai, Nature'(London) 194, 759 (1962). 6 T. Yamanaka and K. Okunuki, Biochim. Biophys. Acta 67, 379 (1963).
[61]
P U R I F I C A T I O N OF E L E C T R O N - T R A N S F E R C O M P O N E N T S
647
duced form ofPseudomonas cytochrome oxidase in turn can be oxidized either by molecular oxygen or by nitrite. Since Pseudomonas cytochrome oxidase is synthesized by the organism only anaerobically in the presence of nitrate or nitrite it was proposed that the enzyme in vivo is really a nitrite reductase. 6 Nonetheless, the term Pseudomonas cytochrome oxidase has been retained. In addition to the oxidase, cytochrome c-551, and azurin, an additional c-type cytochrome, called cytochrome c-556, can be purified from extracts of P. aeruginosa, r Pseudomonas Cytochrome
Oxidase
Ferrocytochrome c-551 + 2H ÷ + 1/202 ~ ferricytochrome c-551 + H20 or
Cuprous azurin + 2H + + 1/202 ~ cupric azurin + H20
Pseudomonas cytochrome oxidase has been isolated in a homogeneous crystalline form from Pseudomonas aeruginosa by Okunuki and his co-workers, 2-4 by Kuronen and Ellfolk, s and by Gudat, Singh, and Wharton. 7 In each case the purified enzyme contains equimolar quantities of heine c and heme dl.
Assay
Method
Principle. Pseudomonas cytochrome oxidase is most conveniently assayed by the spectrophotometric method described by Gudat et al.r The rate of oxidation of Pseudomonas cytochrome c-551 is measured by following the decrease in the absorbance of its a-band at 551 nm. Alternatively, the rate of oxidation of reduced azurin can be measured by observing the increase in its absorbance at 625 nm. Reagents 1. Potassium phosphate buffer, 0.1 M , p H 6.0. 2. Pseudomonas ferrocytochrome c-551: Cytochrome c-551 is purified from P. aeruginosa and is maintained as a stock solution of 200/xM in 0.1 M potassium phosphate buffer, pH 6.0. A few milliliters of the stock solution are reduced by adding a few grains of sodium dithionite; excess dithionite is removed by gently shaking the solution for a few minutes. The solution should be used only on the day it is reduced. r j. C. Gudat, J. Singh, and D. C. Wharton, Biochim. Biophys. Acta 292, 376 (1973). T. Kuronen and N. Ellfolk, Biochim. Biophys. Acta 275, 308 (1972).
648
OTHER MICROBIALELECTRONTRANSPORTSYSTEMS
[61]
3. P s e u d o m o n a s azurin (reduced): Azurin is purified from P. aeruginosa and is maintained as a stock solution of 500 t~M in 0.1 M p o t a s s i u m p h o s p h a t e buffer, p H 6.0. A few milliliters of this stock solution can be reduced and handled exactly as in the case of P s e u d o m o n a s c y t o c h r o m e c-551. 4. Potassium ferricyanide, 0.1 M. 5. E n z y m e : I m m e d i a t e l y before assay, P s e u d o m o n a s c y t o c h r o m e oxidase is diluted to a protein concentration of about 5 mg/ml in 0.1 M potassium p h o s p h a t e buffer, p H 6.0 (0°-4°). Procedure. To each of two 1-ml cuvettes with a 1-cm light path add the following: 0.7 ml of p o t a s s i u m p h o s p h a t e buffer and 0.3 ml of Pseudomonas f e r r o c y t o c h r o m e c-551 or 0.3 ml of P s e u d o m o n a s azurin. The blank cuvette is oxidized with 0.01 ml of p o t a s s i u m ferricyanide. After t e m p e r a t u r e equilibration at 30 ° for 4 min, the reaction is initiated by adding 10/~1 of e n z y m e solution. F o r the oxidation of f e r r o c y t o c h r o m e c-551 the decrease in a b s o r b a n c e is m e a s u r e d at 551 nm; for the oxidation of azurin the increase in a b s o r b a n c e at 625 nm is measured. Definition o f Activity. The activity of P s e u d o m o n a s c y t o c h r o m e oxidase m a y be defined in terms of the first-order velocity constant. Concentration(t~me o) min-' k = 2.3 log Concentration(tim~ 0 + , rain) The specific activity is calculated from the known concentration of c y t o c h r o m e c-551 or azurin and e n z y m e in the a s s a y mixture and the estimated first-order velocity constant. S.A.--
k(concentration of substrate) (concentration of enzyme)
The concentration of c y t o c h r o m e c-551 is calculated on the basis of e,~51,m = 28.3 m M -1 cm-1, 9"10 and that of azurin is based on e62,~nm = 6.95 mM-1 cm-l,10, 11 for the reduced and oxidized forms, respectively. Alternative M e t h o d s o f A s s a y . P s e u d o m o n a s c y t o c h r o m e oxidase activity can also be determined by measuring the uptake of o x y g e n in solution either polarographically or manometrically essentially as described for the assay of m a m m a l i a n c y t o c h r o m e c-oxidase.'2 9 T. Horio, T. Higashi, M. Sasagawa, K. Kusai, M. Nakai, and K. Okunuki, Biochem. J. 77, 194 (1960). 10T. Yamanaka, in "The Biochemistry of Copper" (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), p. 275. Academic Press, New York, 1966. ,1 T. Yamanaka, S. Kijimoto, and K. Okunuki, J. Biochem. (Tokyo) 53, 256 (1963). 1~D. C. Wharton and D. E. Griffiths, Arch. Biochem. Biophys. 96, 103 (1962).
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
649
Assay for Nitrite Reductase Activity. Pseudomonas cytochrome oxidase can be assayed for its nitrite reductase activity using the method of Yamanaka and Okunuki. 6 This spectrophotometric method is essentially the same as that used for measuring oxidase activity except that 0.03 ml of 0.2 M potassium nitrite is included in the assay mixture and the reaction is carried out under anaerobic conditions in Thunberg-type cuvettes. Purification Procedure Two procedures for purifying Pseudomonas cytochrome oxidase are described below. The first procedure has the advantage of conveniently producing fractions containing other oxidation-reaction components of interest that can readily be purified further to homogeneity. All operations are carried out at 0°-4 ° unless specified otherwise. Protein estimations are made by the method of Lowry et al. 13 with bovine serum albumin as the standard.
The Organism and Culture Conditions A culture of Pseudomonas aeruginosa is maintained routinely on an agar slant (2%) containing bouillon-peptone. Slants are stored in the refrigerator, and transfers made to new slants every 2 months. For the large-scale production of cells of P. aeruginosa the organism is grown in three stages after transfer from an agar slant. In the first stage 30 ml of a bouillon-peptone medium (5 g of beef extract, 10 g of peptone, and 2,5 g of NaCI per liter of solution adjusted to pH 7.0 with NaOH) are inoculated with the culture. The flask is shaken for 24 hr at 37 °, then this culture is added to 12 liters of the bouillon-peptone medium and the cells are grown under the same conditions as for the 30-ml volume. For the final stage of cell production the 12 liters of inoculum are added to 190 liters of nitrate medium (10 g of beef extract, 10 g of peptone, 20 g of KNO3, 6.4 g of KH2PO4, 3.6 g of Na2HPO4 per liter of solution) in a large fermentor, and the cells are grown at 37 ° without aeration but with slight agitation. The addition of a few milfiliters of antifoam (Union Carbide Corp., SAG471) is advisable because of frothing from the evolution of gas. When the pH of the culture reaches 8 the cells are harvested using a continuous-flow Sharpies centrifuge. After harvesting, the cell paste is collected and frozen without washing. This paste can then be stored at - 2 0 °. The yield of cells is about 2.3 g per liter of medium. As an alternative to a fermentor, cells can be grown in the nitrate 13 O. H. L o w r y , N. J. R o s e b r o u g h , A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
650
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
medium contained in autoclavable polypropylene bottles of 20-liter capacity. Harvesting of these cells can be accomplished with a refrigerated centrifuge if a continuous-flow Sharples centrifuge is not available.
Preparation of Crude Extract Four hundred grams of the frozen cell paste are thawed and suspended uniformly in 1200 ml of 0.1 M phosphate buffer, pH 6.0, containing l0 mg of deoxyribonuclease (Sigma, type I from bovine pancreas). The suspension is filtered through two layers of cheesecloth, and the cells are broken by passing the suspension through a Gaulin laboratory homogenizer (Gaulin Manufacturing Co., Everett, Massachusetts) at a pressure of 9,000 psi. As the effluent emerges from the homogenizer it is cooled to 5 ° in a cold brine bath and is rehomogenized once. The cooled effluent of the second homogenization is centrifuged for 40 rain at 18,000 g. The brownish supernatant is decanted and stored at 4 °, and the residue is resuspended in 600 ml of the phosphate buffer. This suspension then is rehomogenized in the Gaulin homogenizer. The cooled effluent is centrifuged for 40 rain at 18,000 g, and the supernatant is combined with that of the first centrifugation; the residue is discarded. As an alternative to the Gaulin homogenization an extract may be prepared from an acetone-dried powder of the cells essentially as described by Kuronen and Ellfolk s or by Horio. 2 In this procedure 100 g of acetone-dried cells are stirred in 950 ml of 0.1 M phosphate buffer, pH 7.0, containing 2 mg of deoxyribonuclease, for 3 hr at 5 °. This suspension then is centrifuged for 40 min at 18,000 g and the supernatant is recovered.
Purification Procedure 7 Step 1. Fractionation with Ammonium Sulfate. The combined extract is brought to 40% of saturation by adding solid (NH4)2SO4 (29.0 g/100 ml). This solution is centrifuged for 30 min at 18,000 g. The golden-brown supernatant is decanted and is brought to 95% of saturation by adding solid (NH4)2SO4 (39.8 g/100 ml). This solution is centrifuged for 20 rain at 18,000 g, and the supernatant is discarded. The light tan residue is suspended in 150 ml of distilled HzO and dialyzed (Visking No. 30 dialysis tubing) for 12 hr with two changes against 6 liters of 10 mM phosphate buffer, pH 6.0. Step 2. Sephadex G-IO0 Chromatography. The dialyzed solution is applied to the base of a column (5.0 cm × 100.0 cm) of Sephadex G-100 that is equilibrated with 10 mM phosphate buffer, pH 6.0. The column is
[61]
651
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
eluted with the same buffer by upward flow, and 6-ml fractions are collected by means of a fraction collector. A typical elution pattern, illustrated in Fig. 1, shows the separation of the Pseudomonas cytochrome oxidase from the other oxidation-reduction components contained in the extract. These fractions are retained for further purification.
Step 3. CM-Sephadex C-50 Chromatography. Fractions from the column of Sephadex G-100 that contain cytochrome oxidase activity are combined and applied to the top of a column (2.5 cm × 40.0 cm) of CMSephadex C-50 that is equilibrated with 10 mM phosphate buffer, pH 6.0. The bulk of the greenish-brown Pseudomonas cytochrome oxidase does not adhere to the CM-Sephadex and is eluted with additional equilibrating buffer. Occasionally some of the oxidase may adhere to the column. If this occurs the adhering oxidase may be eluted with 0.1 M sodium phosphate buffer, pH 7.0, after first washing the column with 400 ml of 10 mM sodium phosphate buffer, pH 7.0. It is not usually necessary to use a fraction collector to obtain the oxidase that passes directly through the column. However, in the event that some of the enzyme adheres to the column it is advisable to collect the effluent of the 0.1 M phosphate buffer, pH 7.0, in 6-ml fractions. The effluent from the CM-Sephadex column with oxidase activity is combined and dialyzed (Visking No. 30 dialysis tubing) overnight with one change against 40 volumes of 50 mM Tris-chloride, pH 8.0. @
4
~
P. Cyt Oxidase
~
P. Cu Protein
~ 3
2
a
e m t~
E E
t n
P_= Cyt c SSI
c-SS6 3
II,
Is
0.3
I
2 | 9
0.4
O.S
I 0.6
I 0.7
I 0.8
0.9
1.0
Ve / Vt FIG. 1. Elution pattern of o x i d a t i o n - r e d u c t i o n c o m p o n e n t s ofPseudomonas aeruginosa from a c o l u m n of S e p h a d e x G-100. Elution was m a d e with 10 m M p h o s p h a t e buffer, pH 6.0, and s a m p l e s were collected in 6-ml fractions.
652
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
Step 4. DEAE-Cellulose Chromatography. The greenish brown dialyzate is applied to the top of a column (2.5 cm × 25.0 cm) of DEAEcellulose that is equilibrated with 50 mM Tris-chloride, pH 8.0. The Pseudomonas cytochrome oxidase adheres weakly to the DEAE-cellulose and is eluted with 50 mM Tris-chloride, pH 8.0, in 3-ml fractions. Those fractions with A410 nm : A 2 8 0 nm of greater than 1.0 are combined and dialyzed overnight with one change against 40 volumes of 20 M Trischloride, pH 8.0. Step 5. Gradient Elution Chromatography with DEAE-Celhdose. The dialyzed solution from the preceding step is applied to a column (2.0 cm × 20,0 cm) of DEAE-cellulose that is equilibrated with 20 mM Trischloride, pH 8.0. The green oxidase is eluted by means of a linear gradient of 20 mM to 50 mM Tris-chloride, pH 8.0. The total volume of the gradient buffers is 1000 ml. The effluent is collected in 3-ml fractions and those with A41onm : A 2 8 0 nm of greater than 1.15 are combined. The combined fractions are dialyzed overnight against 40 volumes of 20 mM phosphate buffer, pH 6.5, and concentrated by ultrafiltration (Amicon Diaflo PM-10 membranes or Millipore Immersible Molecular Separator). The results of a typical purification by this procedure are shown in Table I. Step 6. Crystallization. The purified Pseudomonas cytochrome oxidase can be crystallized out of (NH4hSO4. To do this the concentrated solution of enzyme from the preceding step is brought to a slight turbidity
TABLE I PURIFICATION OF Pseudomonas CYTOCHROME OXIDASE BY PROCEDURE I
Fraction Crude extract (NH4)SO4, 40-95% Sephadex G-100 CM-Sephadex DEAE-cellulose Gradient elution on DEAE-cellulose Crystallized
Volume (ml)
Total protein (mg)
Total activity (units)"
Specific activity (units/rag protein)
Purification (fold)
Yield (%)
1690 365 540 595 175 10
27,716 9,672 4,374 3,213 630 98
5,273 2,226 1,793 1,541 724 350
0.19 0.23 0.41 0.48 1.15 3.55
1.0 1.2 2.2 2.5 6. I 18.7
100 42 34 29 14 6.6
68
245
3.60
18.9
4.6
4.5
a Units are expressed as micromoles of cytochrome c-551 oxidized per minute.
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
653
by gradually adding finely powdered (NH4)2SO 4 at room temperature. The turbidity is sedimented out by centrifugation for 10 min at 18,000 g, and the supernatant is allowed to stand at room temperature. In time (124 hr) flat diamond-shaped crystals appear. The crystals can be collected by centrifugation for about 10 min in a clinical centrifuge. The sedimented crystals are dissolved in 20 mM phosphate buffer, pH 6.0.
Purification Procedure H 8 Step 1. DEAE-Celhdose Chromatography. The crude extract is dialyzed for 12 hr against 6 liters of 20 mM potassium phosphate buffer, pH 6.9. Two changes of dialysis buffer are made. The dialyzed extract is applied to the top of a column (5.0 cm × 50.0 cm) of DEAE-cellulose equilibrated with 20 mM potassium phosphate buffer, pH 6.9. The brownish red fraction containing Pseudomonas cytochrome oxidase does not bind to the DEAE-cellulose. Step 2. CM-Celhdose Chromatography. The effluent from the DEAEcellulose column is adjusted to pH 6.4 by adding a suitable amount of 20 mM K2HPO4. After dilution with an equal volume of water, this solution is applied to the top of a column (2.5 cm × 25.0 cm) of CM-cellulose equilibrated with 10 mM potassium phosphate, pH 6.4. The c-type cytochromes in the solution are not retained by the column while the oxidase binds. The column is washed with 10 mM potassium phosphate, pH 6.7, until the effluent is colorless. The Pseudomonas cytochrome oxidase is eluted with 40 mM potassium phosphate, pH 6.9. Those green fractions with oxidase activity are combined and concentrated) by ultrafiltration (Amicon Diaflo PM-10 membrane or Millipore immersible molecular separator). Step 3. Sephadex G-lO0 Chromatography. The concentrated solution is applied to the top of a column (2.5 cm × 95.0 cm) of Sephadex G-100 equilibrated with 10 mM potassium phosphate, pH 6.4. The volume of enzyme applied to the column should not exceed 10 ml. The column is eluted with the equilibrating buffer, and collection of 3-ml fractions is made. Those fractions with A410 nm "A280 nm in excess of 1.0 are combined and concentrated by ultrafiltration as in step 2. Step 4. Crystallization. The concentrated Psendomonas cytochrome oxidase may be crystallized according to the method described in Procedure I. In this case two crystallizations are recommended to achieve maximum purity. The results of a typical purification by this procedure are shown in Table II.
654
O T H E R M I C R O B I A L E L E C T R O N TRANSPORT SYSTEMS
[61]
TABLE lI PURIFICATION OF Pseudomonas CYTOCHROME OXIDASE BY PROCEDURE II" r
Fraction
Volume (ml)
Total protein (rag)
Extract DEAE-cellulose CM-cellulose Sephadex G-100 Crystallized Recrystallized
950 1076 7.6 42 0.8 0.5
5900 1830 66 21 10.9 7.8
Total activity (units)t' 247 162 93 53 35 30
Specific activity (units/mg Purification Yield protein) (fold) (%) 0.04 0.09 1.40 2.52 3.22 3.78
1.0 2.1 33 66 77 90
100 66 38 21 14 12
" Data are from T. Kuronen and N. Ellfolk, Biochem. Biophys. Acta 275, 308 (1972). b Units are expressed as micromoles of cytochrome c-551 oxidized per minute. Properties
Storage and Stability. The purified Pseudomonas c y t o c h r o m e oxidase m a y be stored frozen at - 2 0 ° or below for several w e e k s without significant loss of oxidase or nitrite reductase activity. H o w e v e r , r e p e a t e d freezing and thawing of a sample should be avoided since this results in a noticeable loss of these activities. Purity. The Pseudomonas c y t o c h r o m e oxidase p r e p a r e d by either p r o c e d u r e is h o m o g e n e o u s according to p o l y a c r y l a m i d e gel electrophoresis as well as on the basis of data obtained from sedimentation equilibrium analysis. 7 Composition and Spectral Properties. The absorption spectra of Pseudomonas c y t o c h r o m e oxidase in its oxidized and reduced f o r m s are shown in Fig. 2. In the reduced form the absorption m a x i m a at 549-554 nm, 521 nm, and 418 nm represent the ~-, fl-, and Soret-bands, respectively, of the h e m e c constituent, and those at 620-660 nm and 460 nm represent the a- and Soret-bands, respectively, of the second heine group, h e m e di. T h e s e two heme groups are present in a p p r o x i m a t e l y equimolar amounts and account for o v e r 90% of the metal found in the e n z y m e . The millimolar extinction coefficients of the various m a x i m a are given in Table III. The E P R s p e c t r u m of Pseudomonas c y t o c h r o m e oxidase is shown in Fig. 3. The signals with g values at 2.93, 2.31, and 1.4 can be attributed to the ferric iron of the heine c constituent, and those at 2.45 and 1.71 can be associated with the ferric iron of the h e m e dl in the e n z y m e .
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
!
I
655
I
0.8
0.6 ® u o .-nO4 o
0.2
0
t /
,/
I 400
,\
I 500
Wavelength(nm)
I 600
FIG. 2. Absorption spectra of P s e u d o m o n a s cytochrome oxidase. The e n z y m e was dissolved in 0.1 M p h o s p h a t e buffer, pH 6.5. - - -, T h e oxidized portion; - - , the preparation reduced with Na~2S204.
Those signals with g values at 4.40 and 2.08 are due to contaminating iron and copper, respectively. The ferrous form of the oxidase is diamagnetic and has no EPR spectrum.
Molecular Weight. Pseudomonas cytochrome oxidase has a molecular weight of about 120,000. 7' s The minimal molecular weight on the basis of heme iron content is approximately 34,000. This result suggests the presence of 4 heme groups per 120,000 Mr, or 2 heme c and 2 heme dl groups. T A B L E llI ABSORPTION M A X I M A AND E X T I N C T I O N C O E F F I C I E N T S OF R E D U C E D
Psettdomonas
CYTOCHROME OXIDASE a
Heme group Heme c
H e m e d~
Wavelength (nm)
et' (raM -~ cm -~)
544 549 521 418 655 620 460
29.7 30.2 27.7 182 24.7 24.0 56.0
" Data from T. Horio, T. Higashi, T. Y a m a n a k a , H. Matsubara, and K. Okunuki, J. Biol. C h e m . 236, 944 (1961) and from D. C. W h a r t o n (unpublished).
b Extinction coefficients of the h e m e d~ moiety are very sensitive to pH; those given are for p H 6.5 in 0.I M phosphate.
656
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
l
[61]
gi2.45
H
g=2.931t
gi1.4 ~
Amplification
FIG. 3. Electron paramagnetic resonance (EPR) spectrum of oxidized Pse,domonas cytochrome oxidase. The instrument settings were: power output, 0.3 roW; field modulation, 5 G; temperature, 13°K; microwave frequency, approximately 9250 MHz.
The native oxidase can be dissociated by sodium dodecyl sulfate into two subunits each of about Mr 60,000. The finding '4 that only one Nterminal amino acid, a lysine residue, is present in the protein and that there are only two cysteine residues per Mr 60,000 suggests that the two subunits may be identical in primary structure. Thus, each subunit may contain one heme c group and one heme dl group. Oxidation-Reduction Potentials. The midpoint potentials of the heme c and heme dl constituents of Pseudomonas cytochrome oxidase are +0.288 V and +0.218 V, respectively. '5' ,6 Reaction with Pseudomonas Cytochrome c-551 and Azurin. Pseudomonas cytochrome oxidase accepts electrons from either Pseudornonas cytochrome c-551 or azurin. Maximum activity has been reported at pH 6.0 by Gudat et al. 7 and at pH 5.3 by Kuronen and Ellfolk. 8 With oxygen as the final electron acceptor, the K m for Pseudomonas cytochrome c551 is 10.5 /zM at pH 6.0 and 30 ° while the Km for azurin is 17.2 /xM under the same conditions. Reaction with Molecular Oxygen and Nitrite. Reduced Pseudomonas 14 y. Nagata, T. Yamanaka, and K. Okunuki, Biochim. Biophys. Acta 221, 668 (1970). ,s T. Horio, M. D. Kamen, and H. de Klerk, J. Biol. Chem. 236, 2783 (1961). 16 D. C. Wharton, J. C. Gudat, and Q. H. Gibson, Biochim. Biophys. Acta 292, 611 (1973).
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
657
cytochrome oxidase donates electrons either to molecular oxygen or nitrite. Turnover numbers of 107 mol and 139 mol of cytochrome c-551 oxidized rain -1 tool enzyme -1 with O~ and with nitrite, respectively, have been reported by Kuronen and Ellfolk s at 23 ° and a pH of 6.5. Circumstantial evidence has been presented 17 to indicate that the product of the reduction of 02 in these reactions is H20. The product of the reduction of nitrite is NO on the basis of spectral evidence, is During in vitro reduction of nitrite the enzyme becomes gradually inhibited, with the concomitant formation of a spectral form indistinguishable from that created by adding NO to a sample of the reduced oxidase. Inhibitors. Both cytochrome oxidase and nitrite reductase activities are inhibited 100% by 0.1 mM cyanide. NO and N~- also inhibit these activities. Carbon monoxide (CO--O2, 9: 1) causes a 90% inhibition of oxidase activity but has no inhibitory effect, even at 100% CO, on nitrite reductase activity. Nitrite is a competitive inhibitor of oxygen consumption. 3 Pseudomonas C yt ochr om e c-551 and Azurin F e r r i c y t o c h r o m e c-551 + e
--~ f e r r o c y t o c h r o m e c-551
C u p r i c a z u r i n + e - -~ c u p r o u s a z u r i n
Purification P r o c e d u r e For the purification of Pseudomonas cytochrome c-551 it is convenient to begin with the fractions that elute as a broad band (VeNt = 0.750.95) from the column of Sephadex G-100 (see step 2 of the purification procedure I for Pseudomonas cytochrome oxidase). These fractions contain both Pseudomonas cytochrome c-551 and azurin. Step I. Acid Precipitation. The combined fractions from the Sephadex G-100 column are adjusted to a pH of 3.9 by stirring in a suitable volume of 50 mM acetic acid. The resulting turbid solution is centrifuged for 20 rain at 20,000 g and the clear, grayish supernatant is decanted; the whitish precipitate is discarded. Step 2. CM-Cellulose Chromatography. The clear supernatant from step 1 is mixed with a few milliliters of 0.1 M potassium ferricyanide and is applied to the top of a column (1.5 cm × 25.0 cm) of CM-cellulose that is equilibrated with 50 mM acetate-NH4OH buffer, pH 3.9. The column J~T. Yamanaka, A. Ota, and K. Okunuki, J. Biochem. (Tokyo) 49, 414 (1961). is T. Yamanaka and K. Okunuki, Biochim. Biophys. Acta 67, 394 (1963).
658
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS
[61]
is washed with about 400 ml of the equilibrating buffer, the Pseudomonas cytochrome c-551 is eluted as a red band with 50 mM acetate-NH4OH buffer, pH 4.45. After complete elution of the Pseudomonas cytochrome c-551, the column is washed with an additional 100 ml of the 50 mM acetate buffer, pH 4.45. The blue azurin, which by now has migrated about half-way down the column, is eluted next with 50 mM acetateNH4OH buffer, pH 4.65. At this stage each major component is contaminated by a small amount of the other. Almost all of this contamination can be removed by rechromatography on CM-cellulose.
Step 3. Rechromatography on CM-Cellulose. The solutions of Pseudomonas cytochrome c-551 and of azurin are each adjusted to a pH of 3.9 by adding a suitable volume of 50 mM acetic acid. Each solution is then added to the top of separate columns (1.5 cm x 25.0 cm) of CMcellulose equilibrated with 50 mM acetate-NH4OH buffer, pH 3.9. The Pseudomonas cytochrome c-551 in the one column is eluted using 50 mM acetate-NHaOH buffer, pH 4.40. The red effluent is dialyzed with two changes for 12 hr against 40 volumes of 0.1 M phosphate, pH 6.0, and concentrated by membrane ultrafiltration (Amicon Diaflo PM-10 or Millipore immersible molecular separator). The second column of CM-cellulose, adsorbing the blue azurin, first is washed with 400 ml of 50 mM acetate-NH4OH buffer, pH 4.45. The azurin is eluted as a blue band using 50 mM acetate-NH4OH buffer, pH 4.65. The blue effluent is dialyzed with two changes for 12 hr against 40 volumes of 0.1 M phosphate buffer, pH 6.0, and concentrated by membrane ultrafiltration as in the case of the cytochrome c-551. Step 4. (a) Crystallization of Pseudomonas Cytochrome c-551. The solution of Pseudomonas cytochrome c-551, concentrated in the preceding step, is first assayed for its absorbance at 551 nm using a suitably diluted aliquot reduced with a few grains of sodium dithionite. The A551nm c m - 1 of the undiluted solution must be at least 30 before crystallization is attempted. If the concentration is of sufficient strength, a fine powder of (NH4)2SO4 is added in small portions at room temperature until a slight turbidity appears. The pH of the solution is maintained at about 7.5 by the addition of dilute NH4OH. The turbid solution is centrifuged for 10 rain at 10,000 g at room temperature, and the clear red supernatant is decanted. This supernatant is reduced by adding a few grains of sodium dithionite. This solution becomes turbid and upon standing overnight at room temperature is centrifuged for 10 min in a clinical centrifuge at room temperature to collect the crystals. The supernatant, which contains about 30% of the original Pseudomonas cytochrome c-551, is decanted and should be retained. The fine needle-shaped crystals can be dissolved
[61]
PURIFICATION OF ELECTRON-TRANSFER COMPONENTS
659
in a small amount of cold H20 and can be rid of residual (NH4)2804 by passing the solution through a small column (1.0 c m x 15.0 cm) of Sephadex G-25 equilibrated with 0.1 M phosphate buffer, pH 6.0.
(b) Crystallization of Pseudomonas Azurin. The absorbance at 625 nm of the azurin obtained in step 3 is determined using a suitably diluted aliquot of the oxidized preparation. If the A625nm of the undiluted solution is at least 10, then the crystallization procedure can be commenced. To do this a fine powder of (NH4)2SO4 is added gradually until a slight turbidity is observed. The pH is maintained at about 7.0 during this period by adding dilute NH4OH from time to time. After 2 hr the slightly turbid solution is centrifuged for 10 min at 10,000 g at room temperature, and the clear blue supernatant is decanted. A small amount of finely powdered (NH4)zSO4 is added to the supernatant solution until a slight turbidity begins to form. This solution is allowed to sit at room temperature for a day or two, during which time dark blue, needle-shaped crystals form. The crystals are harvested by centrifugation for 10 min in a clinical centrifuge at room temperature. The blue supernatant can be retained. The crystalline pellet is dissolved in a small volume of cold H20 and separated from residual (NH4)2SO4 by chromatography on a small column (1.0 x 15.0 cm) of Sephadex G-25 equilibrated with 0.1 M phosphate buffer, pH 6.0.
Properties of Pseudomonas Cytochrome c-551 and Azurin
Storage and Stability. Buffered solutions of Pseudomonas cytochrome c-551 and azurin can be stored at - 2 0 ° or below for months without noticeable deterioration. Lyophilized samples kept for up to 2 years at - 2 0 ° or below have retained their normal reactivities with Pseudomonas cytochrome oxidase and exhibit no alteration of their spectral properties. Purity. Samples of both Pseudomonas cytochrome c-551 and azurin that have undergone two chromatographic steps on CM-cellulose followed by crystallization exhibit only a single protein-staining band following polyacrylamide gel electrophoresis. Spectral Properties. The absorption spectra of Pseudomonas cytochrome c-551 in its ferric and ferrous forms are shown in Fig. 4. The ferrous form is characterized by absorption maxima in the visible region at 551,521, and 416 nm. On the basis of the iron content E551n m has been calculated to be 28.3 mM -1 cm -1 for the reduced form. 9"lo The absorption spectra of Pseudomonas azurin in its cupric and cuprous forms are
660
OTHER MICROBIAL ELECTRON TRANSPORT SYSTEMS 0.75
t
[61]
u
=~o.so i,~_ ,< 0.25
0
I I 400 500 Wavelength (nm)
600
FIo. 4. Absorption spectra of Pseudomonas cytochrome c-551. The protein was dissolved in 0.1 M phosphate buffer, pH 6.5. - - -, The oxidized portion; , the preparation reduced with Na25204.
illustrated in Fig. 5. The cupric form shows a broad absorption maximum near 625 nm and a very broad and very small absorption between 325 nm and 450 nm. The reduced or cuprous azurin has no absorption maxima in the visible region. On the basis of the copper content %25nm has been calculated to be 6.95 mM -1 cm -1 for the cupric protein. 10, 11
Molecular Weight. On the basis of its iron content, the minimal molecular weight of Pseudomonas cytochrome c-551 has been calculated to be 8100, and a value of 7600 has been obtained from sedimentation velocity data. 9' 10 These results indicate the presence of one heme group per protein molecule. The molecular weight of Pseudomonas azurin has been determined
I
I
I
/f ~',\\
0.6
iI
i0.4 .-0
\',
/ /
~=)
ii
~[ 0.2
\\
\\ \x
/" i//
~00 I
x~
600 I
Wavelength
760
(nm)
~do
F I G . 5 . Absorption spectra ofPseltdornonas azurin. The protein was dissolved in 0 . 1 M phosphate buffer, pH 6 . 5 . - - -, The oxidized portion; - - , the preparation reduced with
Na~25204.
[61]
PURIFICATION OF ELECTRON-TRANSFERCOMPONENTS
661
to be about 17,400 from sedimentation velocity data and about 16,600 on the basis of copper content.l°' 11 These data support the conclusion that each protein molecule contains one atom of copper.
Oxidation-Redaction Potentials. The oxidation-reduction midpoint potentials of Pseudomonas cytochrome c-551 and azurin at pH 6.4 have been measured as +0.286 V and +0.328 V, respectively. 1° Electron-Transfer Reactions. Both reduced Pseudomonas cytochrome c-551 and azurin donate electrons to Pseudornonas cytochrome oxidase. Both proteins in their oxidized forms are capable of accepting electrons from NADH by means of a soluble Pseudomonas NADH dehydrogenase and certain quinones.19 In addition, the Pseudomonas cytochrome c-551 and azurin are able to transfer electrons between themselves. 2° A number of reducing agents, including N a 2 5 2 0 4 and ascorbic acid, reduce both proteins. ~9D. C. Wharton, unpublished results. ~0 E. Antonini, A. Finazzi-Agro, L. Avigliano, P. Guerrieri, G. Rotilio, and B. Mondovi, J. Bbd. Chem. 245, 4847 (1970).
