ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 19’7, No. 1, October I, pp. 1-9, 1919
Isolation
LAURIE Departments
and Characterization of Cytochrome from Tetrahymena pyriformis KILPATRICK’
AND
MARIA ERECIfiSKA
of Biochemistry and Biophysics and Pharmacology, Medical School, Philadelphia, Pennsylvania Received
d
University 19101,
of Pennsylvania
March 5, 1979; revised May 30, 1979
Partially purified cytochrome d free of other cytochrome components has been isolated using detergent (Triton X-100) and salt (KCl). The isolated cytochrome d, has an absorption maximum at 615-617 nm and is capable of oxidizing the reduced cytochrome cjsa of tetrahymena. Potentiometric titrations show that cytochrome d like cytochrome aa has a twocomponent structure with half-reduction potentials of 0.17 and 0.32 V. EPR studies of aerobic cytochrome d show low spin ferric heme resonances at g = 2.96 and g = 2.26 and a g = 2.0 signal characteristic of oxidized copper. EPR studies on oriented multilayers of partially purified cytochrome d show that the heme seen in the fully oxidized preparation is oriented in such a way that the angle between the heme normal and the membrane normal is approximately 90”.
It has been known since the early studies versity of Pennsylvania) was grown and harvested as of Warburg and his co-workers (1, 2) and described previously (6) and mitochondria were isoKeilin (3) that pigments other than cyto- lated according to the method given in Ref. (6). The chrome uu3 can function as the terminal oxi- final mitochondrial pellet was suspended at a protein concentration of approximately 40 mg/ml in 0.25 M dases in bacterial systems. One of these al- sucrose-10 mM KCl-5 mM Mops*-0.2 mM EDTA conternate oxidases, cytochrome d (a,), was taining 0.2% bovine serum albumin and 10% dimethyldiscovered by Yaoi and Tamiya (4) in Esche- sulfoxide (pH 7.4), quickly frozen in liquid nitrogen, richia coli and Shigella dysenteriae and fur- and stored below -18°C. ther investigated by Keilin (5) in Axotobacter. We have shown previously (6) that cyto- Zsolation and Partial PuriIcation of chrome d is the terminal oxidase in the ciliCytochrome d ate protozoan, Tetrahymena pytiformis. In The mitochondrial suspension was thawed, diluted this paper we describe an isolation tech- approximately 15-fold in 0.25 M sucrose-O.01 M phosnique which yields a partially purified active phate buffer (pH 7.2), and centrifuged at 10,000 rpm cytochrome d. The properties of cyto- for 10 min. The pellet was resuspended in 0.25 M suchrome d as well as its orientation with re- crose-0.01 M phosphate buffer (pH 7.2) at a protein spect to the plane of the membrane are dis- concentration of 30 mgiml and treated with 10% Triton cussed and compared to those of cytochrome X-100 (final concn = 0.7 mg/mg protein) and solid KC1 (final concn = 1 M). The pH was maintained at 7.4 by addition of 0.5% ammonia in water. The mixture was stirred for 20 min at 4°C and centrifuged for 45 min at 32,000 rpm in a Beckman Model L5-50 ultracentrifuge. After centrifugation, several distinct fractions were obtained. The top, light pink supernatant layer
aa3. METHODS AND MATERIALS Z’etrahymena pytiformis, strain ST, (kindly provided by Dr. Y. Suyama, Department of Biology, Uni-
(about 90% of the total supernatant)
contained soluble
mitochondrial proteins and the remaining cytochrome
’ To whom correspondence should be directed. Supported by USPHS HL 18-708 and GM 12202. M.E. is an Established Investigator of the American Heart Association.
2 Abbreviations used: Mops, 3-(N-Morpholino)propanesulfonic acid. 1
WO3-986~79/11099&09$02.00/0 Copyright 0 1979 by AcademicPress, Inc. AU rights of reprodn&ion in any form reserved.
2
KILPATRICK
AND ERECINSKA
c and b,, pigment. It was pipetted off and discarded. The remaining bottom, supernatant layer was dark red and appeared quite viscous. It contained the bulk of the cytochrome be, complex. The pellet consisted of a tightly packed black sediment surrounded by a loosely packed greenish-brown layer which contained cytochromed. Since it was difficult to separate the be, complex from the oxidase layer at this stage, the dark red supernatant layer and the bottom greenish-brown sediment were suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) and centrifuged for 30 min at 32,000 rpm. The tightly packed black pellet was discarded. Good separation was achieved; the cytochrome d formed a tight greenish brown pellet whereas the bc, complex remained soluble in the supernatant. The supernatant was discarded and cytochrome d was resuspended in a minimal amount of 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2).
Analytical
Methods
Absolute and dvference spectra were obtained using a Johnson Foundation dual wavelength scanning spectrophotometer adapted for use with a PDP-11 computer. The detailed conditions are given in the figure legends. Cytochrome d activity. The activity of partially purified cytochrome d was determined by measuring the rate of oxidation of isolated reduced T. pyriformis cytochrome c5= by the solubilized cytochrome d fraction. Isolated cytochrome es53(7) was reduced with sodium dithionite and filtered through a Sephadex G-25 column to remove excess dithionite. The rates of oxidation were measured in a dual wavelength spectrophotometer by monitoring the decrease in absorption at 553540 nm at 22°C. Cytochrome d activity was also measured in the presence of two inhibitors, potassium cyanide and sodium sulfide. The experimental conditions were the same as described above (see figure legends for the details). Determination of copper and heme contents. The copper content of isolated cytochrome d was determined by two different methods. In the first one, copper was measured calorimetrically by the biquinoline method of Brumby and Massey (8) using cupric sulfate as the standard. In the second, the oxidized copper signal at g = 2.0 was quantitated by double integration using a Varian E-109 spectrometer equipped with a Model 1074 instrument computer (Nicolet Instt-ument Corporation) and compared with double integrated signals of copper-EDTA standards at concentrations between 10 pM and 1 mM. All measurements were made using nonsaturating microwave powers at 18 K. An approximation of the heme content was made by comparison of the low spin ferric heme resonance of cytochrome d at g = 2.96 with the low spin ferric heme resonance at g = 3.0 of mammalian cytochrome aa prepared from pigeon breast mitochondria.
Protein Determination. Proteins were assayed by the biuret method (9) using crystalline albumin (Sigma Chemical Co.) as a standard. Potentiometric titrations were carried out by simultaneous measurements of absorbance (dual wavelength spectrophotometer) and the oxidation-reduction potentials (redoxostat) at 22°C. The redoxostat used is similar to that described by Swartz and Wilson (10) with modifications by Wilsonet al. (11). The redoxostat utilizes four electrodes; the measuring electrodes are glassy carbon with a standard calomel reference, the working electrode is glassy carbon with Ag-AgCl counter electrode. Methyl viologen (Sigma Chemical Co.) was used as the primary electron acceptor for the working electrode, while ferrocenyl methylitrimethyl ammonium iodide (ICN Pharmaceuticals, Inc.) and ferricyanide acted as electron donors. The oxidase (approx. 15-20 mg protein/ml) was suspended in 0.25 M sucrose-O.01 M Pi, pH 7.2 and phenazine methosulfate (Sigma Chemical Co.) and diaminodurene (Aldrich Chemical Co.) were used as redox mediators. The results are presented graphically as the logarithm of the ratio of oxidized to reduced forms of the cytochrome (abscissa) as a function of the oxidationreduction potential (ordinate). The titration curves were separated into individual components as described by Wilson and Dutton (12)and Duttonet al. (13). Electron
paramagnetic
resonance
measurements.
EPR spectra were measured using a Varian E-109 spectrometer equipped with an Air Products LTD 3-110 liquid helium cryostat. Preparation oforiented cytochrome d. Oriented multilayers of tetrahymena cytochrome d were prepared as deseribed by Erecinska et al. (14). It was found that thin samples (-11 mg protein for a l-cm-diameter specimen) required less time for partial dehydration and produced better oriented specimens.
RESULTS
Isolation
of Cytochrome
d
The procedure described above provides a method for isolating cytochrome d of T. pytiformis. Although the method appears simple, the main problem lies in that cytochrome d is highly unstable and denatures easily (15). Therefore standard methods used for isolating cytochrome aa which utilize extensive fractionation in the presence of salt and detergent cannot be applied to isolate an active cytochrome d. In developing the isolation procedures we have found that certain precautions must be taken with the storage of the tetrahymena mitochondria to insure isolation of a spectrally un-
ISOLATION
AND CHARACTERIZATION
modified enzyme. The mitochondria must be rapidly frozen in liquid nitrogen in the presence of dimethyl sulfoxide to prevent formation of ice crystals which disrupt the mitochondrial structure. Moreover, they should be stored either in liquid nitrogen or at below -20°C in a freezer which maintains a constant low temperature and does not have a defrost cycle. Better yields of cytochrome d were consistently obtained from mitochondria which were stored for less than a week. The first centrifugation after thawing of the mitochondria can usually provide an indication as to the yield and quality of cytochrome d. If the supernatant is pink due to the liberation of the loosely bound cytochrome es53it has been our experience that subsequent cytochrome d yield will be very small and the enzyme will not be active. When the precautions described above were taken, the procedure yielded in our hands reproducible and reliable results. An overall yield of 84% was obtained by calculating the recovery of heme d in the oxidase fraction from a known concentration of mitochondria. The purification was threefold as measured by the increase of heme d content per milligram protein. This
OF CYTOCHROME
d
3
low purification value is due to the fact that the partially purified cytochrome d, although deficient in other cytochromes, contains nevertheless relatively high amounts of colorless proteins. Attempts of further purification were unsuccessful because any additional manipulation led to modification and inactivation of this enzyme. Characteristics
Cytochrome
of the Isolated d
Figure 1 (I) shows the spectral properties of the isolated cytochrome d in the visible region. The sharp asymmetric peak at 61’7 nm is due to cytochrome d whereas a small peak at 560 nm may be attributed to contamination with the cytochrome b-c 1 complex (less than 10%). The absorption spectra of the isolated oxidized and reduced cytochrome d are presented in Fig. 1 part II, A and B, respectively. The Soret absorption maximum of oxidized cytochrome d is at 420 nm while the reduced form exhibits a Soret peak at 445 nm. The difference spectrum for the isolated cytochrome d in the Soret region (reduced-oxidized) is shown in Fig. 1, part
FIG. 1. The absolute and differenee spectra of isolated cytochrome d in the (Yand Soret region. The absolute and difference spectra were obtained using a PDP-11 computer operated scanning dual wavelength spectrophotometer. (I) The difference spectrum of isolated cytochrome d in the a and p region. Cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer at pH 7.2 at a protein concentration of 0.29 mg/ml. The spectrum of the fully oxidized sample was measured and stored in the computer memory as the baseline. The sample was reduced with dithionite and measured as (dithionite-reduced) - oxidized. The reference .wavelength was 575 nm. (II) The absolute spectra of isolated cytochrome d in the Soret region. Cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 0.14 mgimi. The reference wavelength was 460 nm. The spectrum of the buffers alone were measured and stored in the eomputer memory as the baseline. (A) Fully oxidized - buffer. (B) (Dithionite-reduced) - buffer. (C) (Dithionite-reduced + CO) - buffer. (III) The difference spectra of isolated cytochrome d in the Soret region obtained from the absolute spectra shown in part II. (A) (Dithionite-reduced)-oxidized. (B) (Dithionite-reduced + CO)-oxidized.
4
KILPATRICK
AND ERECINSKA
III, trace A and has an absorption maximum at 444 nm.
dized and reduced cytochrome d in the Soret region of the spectrum. Although the spectral changes are small, they are clear enough to conclude that cyanide binds to both the reduced and the oxidized forms of the enzyme.
Ligand Binding The effects of ligand binding on the absorption spectra of isolated cytochrome d were studied using the following ligands: carbon monoxide, nitric oxide, and cyanide. In Fig. 1 (parts II and III) the absolute and difference spectra of the CO-cytochrome d complex are shown in the Soret region. The absolute spectrum of the COcompound was obtained by bubbling the dithionite-reduced sample with CO gas for a period of 3 min. It can be seen that the CO-difference spectrum [(dithionite reduced + CO) - (dithionite reduced)] of cytochrome d exhibits an absorption maximum at around 428 nm. Figure 2, part I (trace C) shows the absolute spectrum of the NO-cytochrome d complex in the Soret region obtained upon the addition of sodium nitrite to the dithionite-reduced sample. A broad peak is observed with an absorption maximum at around 430 nm. Figure 2, parts II and III, shows the effect of cyanide addition to oxi-
Copper and Heme Determinations Previous preliminary EPR studies (‘7) suggested that cytochrome d, like cytochrome aa3, contains copper. Measurements of the copper content calorimetrically on partially purified preparations showed it to be 1.48 2 0.01 nmol copperlmg protein. The heme content of the preparation was calculated making the assumption that the extinction coefficient for cytochrome d at 617-640 nm was 25 mM-’ cm-‘. [It should be mentioned that attempts were made to convert heme d to its alkaline hemochromogen (6) and thereby determine its concentration. However, this led to modification of the heme into a compound for which the spectral properties are not known.] Calculation of the heme content using the above extinction coefficient gave a value of 0.85 4 0.11 nmol heme/mg protein, i.e., -0.5 m
400
420
440
460 Alnm)
480
500
400
420
440 hlnml
460
480 -a
400
440
480
h(nm)
FIG. 2. The effects of cyanide and NO addition on the absolute spectra of isolated cytochrome d in the Soret region. The absolute spectra were obtained using a PDP-11 computer-operated scanning dual wavelength spectrophotometer with a reference wavelength at 460 nm. Isolated cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 0.14 mg/ml. The spectrum of the buffer alone was measured and stored in the computer memory as the baseline. (I) (A) Fully oxidized sample - buffer. (B) (Dithionite-reduced sample) - buffer. (C) (Dithionite-reduced sample + NO) - buffer. The sample was reduced with a few crystals of sodium dithionite and 50 mM sodium nitrite was added. (II) (A) Fully oxidized sample - buffer. (B) (Fully oxidized sample + 5 mM KCN) - buffer. (C) (Dithionite-reduced sample with KCN present from B) - buffer., (III) (A) Fully oxidized sample - buffer. (B) (Dithionite-reduced sample) - buffer. (C) (Dithionite-reduced sample + 1 mM KCN) - buffer.
ISOLATION
AND CHARACTERIZATION
OF CYTOCHROME d
5
heme to 1 copper. However, if cytochrome d contains two hemes [as shown in the po-
tentiometric titrations in Ref. (6) and Fig. 31 and if by analogy with cytochrome uu3 only one of the hemes is responsible for the absorption properties in the visible region, then the heme to copper ratio would be close to unity. These findings were substantiated by results obtained with EPR spectroscopy. Double integration of the copper signal in the oxidized preparation of cytochrome d yielded a value of approximately 0.7 nmol of copperlmg protein which is about one-half the value obtained calorimetrically. This suggests that only one of the two coppers of cytochrome d is detectable by EPR measurements. The heme content of isolated cytochrome d was estimated by comparing the low spin ferric heme resonance at g = 2.96 with the g = 3.0 signal of isolated cytochrome ua3 whose heme content has been calculated from optical absorbance measurements [millimolar extinction coefficient at 605 nm (reduced) - 630 nm (oxidized) = 26.4 cm-‘]. Such estimates gave a value of approximately 0.6 nmol of cytochrome d heme/mg protein. Therefore, the EPR measurements yielded a heme to copper ratio of 1 to 1. Since only one of the coppers of cytochrome d is detectable by EPR, it is reasonable to conclude that only one of the hemes is detectable. This is similar to the situation seen with mammalian oxidase. Activity
of Zsolated Cytochrome
d
In order to establish that isolated cytochrome d is still functional, its ability to oxidize reduced tetrahymena cytochrome c5% was measured and compared with values obtained with tetrahymena oxidase in intact mitochondria (7). Isolated cytochrome d was able to oxidize reduced tetrahymena cytochrome es53at rates comparable to those seen in intact mitochondria and as shown in Fig. 3 part I, the rate of oxidation increased -proportionally with increasing concentrations of cytochrome d. By extrapolation from the initial velocity rates of oxidation of reduced cytochrome css3,double-reciprocal plots were obtained which gave a K, ‘of approximately 20 ~.LM
FIG. 3. Activity of isolated cytochrome d. Part (A). Reduced tetrahymena cytochrome css3(24 pM) was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at room temperature. The reaction was started by the addition of various concentrations of isolated cytochrome d (A: 0.24 mg protein, B: 0.48 mg protein, and C: 0.96 mg protein). The rate of oxidation was measured spectrophotometrically at 553-540 nm. (Part B). The effects of cyanide and sulfide on the activity of cytochromed. The conditions are the same as described above using 24 pM cytochrome cAa3and 0.96 mg protein cytochrome d.
(not shown). This value is similar to the K, of 5-10 &&M obtained with intact mito-
chondria using the same procedure. To further establish that cytochrome d has not undergone modification during isolation, the effect of inhibitors on the rate of oxidation was measured. Part II of Fig. 3 shows that the addition of cyanide and sulfide completely inhibits oxidation of reduced cytochrome es53by cytochrome d. In intact mitochondria using NADH as substrate, it was found that 1 mM KCN inhibits oxidation of this substrate by 90%, 50 mM Na,S by 85%, 50 mM hydroxylamine by 65%, and 50 mM azide by 40% (data not shown). Therefore, it appears that cytochrome d isolated by this method has not undergone any severe modification. Furthermore, it shows
6
KILPATRICK
AND ERECINSKA
the same response to inhibitors as the membrane-bound enzyme. Oxidation-Reduction Potentials Isolated Cytochrome d
of
-
/-Lfr ,-?9h
I
’ h------
Anaerobic potentiometric titrations of suspensions of isolated cytochrome d were carried out as described in methods and the results are shown in Fig. 4. The titration FIG. 5. The EPR spectra of partially purified yields a sigmoidal curve which can be re- aerobic cytochrome d. Cytochrome d was suspended solved into two components with n = 1 and in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at half-reduction potentials of 0.170 and 0.320 a protein concentration of 22 mgiml and frozen in liquid V at pH 7.2 and 22°C. These midpoint nitrogen in quartz EPR tubes. EPR frequency of 9.106 potential values are close to those obtained GHz, microwave power 5 mW, and temperature 15°K. from titrations of cytochrome d in intact Scanning time: 4 min, time constant: 1s. tetrahymena mitochondria (6) and submitochondrial particles (7). plane of the membrane at various angles to the direction of the magnetic field are shown EPR Spectra of Isolated Cytochrome d in Fig. 6. The magnitudes of the compoThe EPR spectrum of partially purified nents of the g tensor exhibit dependence on the direction of the applied magnetic field. aerobic cytochrome d sample is shown in The lowest field signal at g = 2.94 is maxiFig. 5. The low spin ferric heme signals mal when the planes of the oriented multiat g = 2.96 and g = 2.27 are due to cytolayers are parallel to the magnetic field and chrome d. A prominent g = 2.0 signal is minimal when the planes of the membranes characteristic of oxidized copper. are normal to the magnetic field. The two maxima of the g = 2.94 signal which are EPR Spectra of Oxidized Cytochrome d in seen in the plot of signal magnitudes at variOriented Multilayers of Partially ous angles (Fig. ‘7) are exactly 180” apart. Purified Enx yme The g = 2.26 signal exhibits opposite orienThe EPR spectra of the oriented multi- tation. It is maximal when the plane of the layers of oxidized cytochrome d with the membrane is parallel to the direction of the magnetic field. The maxima are also 180” apart and 90” out of phase with the g = 2.94 resonance. DISCUSSION
The results presented in this work offer a simple and quick procedure for the preparation of cytochrome d essentially free of other cytochrome components. This is, to our best knowledge, the first successful attempt of isolation of this bacterial-type oxidase. The isolated cytochrome d has the same FIG. 4. Potentiometric titration of cytochrome d. spectral properties as does the hemeprotein Cytochrome d was titrated as described under Matein intact T. pytiformis mitochondria and rials and Methods by following absorbance changes at catalyzes the oxidation of reduced cyto617-575 nm. Cytochrome d was suspended in 0.25 M chrome cSS3by oxygen at a comparable sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 15 mgiml. The redox mediators used velocity. Two components are observed during anaerobic potentiometric titrations are listed under Materials and Methods. The different of partially purified cytochrome d with halfsymbols represent oxi’dative and reductive titrations. Solid lines represent theoretical n = 1. reduction potential values slightly lower
ISOLATION
AND CHARACTERIZATION
OF CYTOCHROME d
Tetrahymeno Pyrlformls Oxldase
nrg=226
3
40”
2000
II
2200
/
2400
2600
2800
300
Magnetic Field (Gauss1
MAC-498
FIG. 6. EPR spectra of oxidized cytochrome d in oriented multilayers of the “membranous” enzyme at various angles between the direction of the magnetic field and the normal to the membrane plane. The oriented l-cm multilayer was formed from 11 mg protein of cytochrome d and partially dehydrated for approximately 48 h at 90% relative humidity and 4°C. The partially hydrated multilayer with its supporting mylar was cut into 2-mm strips and inserted into a quartz capillary tube and frozen in liquid nitrogen. EPR frequency: 9.11 GHz, microwave power: 10 mW, sample temperature 6.5”K, scanning time: 2 min, time constant: 0.25 s, and modulation amplitude: 2.5 x 10 G;
than those obtained for the native system (0.32 vs 0.345 V and 0.17 vs 0.245 V). This shift in the half-reduction potential values may be caused by the presence of a detergent which modifies the environment of the heme. Such changes have been observed in other preparations of mitochondrial complexes in the presence of detergents (16). The two-component nature of cytochrome d may be, like that of cytochrome aa3, the intrinsic property of this enzyme. Cytochrome d is capable of binding carbon monoxide and forming a characteristic reduced cytochrome d-CO complex. The EPR studies on isolated cytochrome d showed the presence of a low spin ferric heme which exhibits resonances at g = 2.94-2.96 and g = 2.26. The magnitude of the highest field g signal was too small to determine its position with sufficient ac-
Tefrahymena Pyriformls Oxldase g’226
60? 7.4 50E 3
,_
l=“’
\y , , I
r, \
:
0 20”
60’
100” Angle
140”
180’
220”
MM - 49,
FIG. 7. Plot of relative EPR signal intensities for oxidized cytochrome d in oriented multilayers as a function of the angle between the magnetic field direction and normal to the plane of the membrane. The EPR spectra were recorded under conditions specified in Fig. 6.
8
KILPATRICK
AND ERECINSKA
curacy. In addition, the presence of a pronounced g = 2.0 signal characteristic of oxidized copper, suggests that cytochrome d, like cytochrome aa3 is a heme-copper protein. This conclusion is supported by chemical analysis which shows copper to be present in the preparation in amounts comparable to those of the heme( Successful preparation of hydrated oriented multilayers from partially purified cytochrome d permitted the determination of the orientation of the heme chromophore(s) with respect to the plane of the multilayers. Since the heme structure of cytochrome d as well as the three-dimensional configurations of the enzyme are not known at the present time, the interpretation which we offer involves certain assumptions and relies on the information obtained from single crystal analysis of cytochrome c (17) and low spin ferric derivatives of myoglobin (18,19). The lowest field g resonance at g = 2.942.96 is maximal when the planes of the multilayers are parallel to the magnetic field (Figs. 6 and ‘7). Thus, if one assumes that this resonance arises from the g, component of the g tensor (i.e., the one which is approximately normal to the plane of the heme (20)) the conclusion can be drawn that the oxidized heme of cytochrome d is oriented normal to the plane of the multilayer. This also means that the oxidized heme of cytochrome d is similarly oriented to that of cvtochrome aan. Such an orientation was measured for the latter heme in oriented multilayers from a “membranous” form of the isolated enzyme (21, 22) in oriented multilayers of mitochondrial membranes (23), and their fragments (24), and in oriented multilayers from a bacterium P. denitrificans
(25).
The g = 2.26 resonance is maximal when the planes of the membranes are normal to the magnetic field. Assuming that this resonance arises from the gl, component of the g tensor (i.e., one of the two principal components of the g tensor which lie in the plane of the heme (20)) one can conclude that the g, axis is oriented parallel to the planes of the membranes. This orientation of the gU axis in fully oxidized cytochrome d is different from that of cytochrome aa3 from pigeon breast mitochondria and P. denitrif-
icans (25). In view of the very limited knowledge of the chemistry of cytochrome d it is difficult to draw any further conclusion from this finding. In summary, cytochrome d has been partially purified from mitochondria of T. pyriforrnis and its spectral and thermodynamic properties have been measured. The orientation of the heme of cytochrome d with respect to the planes of the oriented multilayers has been determined. The availability of yet another terminal oxidase may prove helpful in our understanding of the operation of cytochrome oxidases. REFERENCES 1. WARBURG, O., AND NEGELEIN, E. (1929) Biothem. Z. 214, 64-100. 2. WARBURG, O., NEGELEIN, E., AND HAAS, E. (1933) Biochem. Z. 266, 1-8. 3. KEILIN, D. (1927) C. R. Sot. Biol. Paris 96, sp39-Sp68. 4. YAOI, H., AND TAMIYA, H. (1928) Proc. Imp. Acad. Japan 4, 436-439. 5. KEILIN, D. (1933) Nature (London) 132, 783. 6. KILPATRICK, L., AND ERECI~~SKA, M. (1977) Biochim. Biophys. Acta 460, 346-363. 7. KILPATRICK, L., AND ERECI~SKA, M. (1977) Biochim. Biophys. Acta 462, 515-530. 8. BRUMBY, P. E., AND MASSEY, V. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 463-474, Academic Press, New York. 9. GORNALL, A. B., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 10. SWARTZ, D. B., AND WILSON, G. S. (1971) Anal. Biochem. 40, 392-400. 11. WILSON, D. F., ERECI~~SKA, M., SANDLER, S., AND NELSON, D. (1979) in International Symposium on Oxidases and Related OxidationReduction Systems, III (King, T. E., Mason, H., and Morrison, M., eds.), Albany, N.Y., in press. 12. WILSON, D. F., AND DUTTON, P. L. (1970) Biothem. Biophys. Res. Commun. 39, 59-64. 13. DUTTON, P. L., WILSON, D. F., AND LEE, C. P. (1970) Biochemistry 9, 5077-5082. 14. ERECI~~SKA, M., BLASIE, J. K., AND WILSON, D:F. (1977) FEBS Lett. 76, 235-239. 15. LLOYD, D., AND CHANCE, B. (1972) Biochem. J. 128, 1171-1182. 16. LEIGH, JR., J. S., AND ERECI~~SKA, M. (1975) Biochim. Biophys. Acta 387, 95-106. 17. MAILER, C., AND TAYLOR, C. P. S. (1972) Canad. J. Biochem. 50, 1048-1055. 18. HELCKE, G. A., INGRAM, D. J. E., AND SLADE, E. F. (1968) Proc. Roy. Sot. B 169, 275-288.
ISOLATION AND CHARACTERIZATION OF CYTOCHROME d 19. H0~1,H.(1971)Biochim.Biophys. Acta251,227235.
9
23. ERECI~~SKA, M., WILSON,D. F., AND BLASIE, J. K. (1978)Biochim.Biophys. Acta 501,63-‘72.
20. GIBSON,J.F., ANDINGRAM,D.J. E. (1957)Na24. BLUM, H., HARMON, H. J., LEIGH, J. S., ture (London) 180,29. SALERNO,J. C., ANDCHANCE,B. (1978)Bio20. BLASIE,J.K.,ERECII;JSKA,M.,SAMUELS,S.,AND chim. Biophys. Acta 502, l-10. LEIGH,JR., J. S. (1978)Biochim. Biophys. Acta 501, 33-52. 25. ERECI~SKA,M., WILSON,D. F., AND BLASIE, 22. ERECI~~SKA, M., WILSON,D. F., AND BLASIE, J. K. (1979)Biochim. Biophys. Acta 545, 352J. K. (1978)Biochim. Biophys. Acta 501,53-62. 367.
Vol. 19’7, No. 1, October I, pp. 1-9, 1919
Isolation
LAURIE Departments
and Characterization of Cytochrome from Tetrahymena pyriformis KILPATRICK’
AND
MARIA ERECIfiSKA
of Biochemistry and Biophysics and Pharmacology, Medical School, Philadelphia, Pennsylvania Received
d
University 19101,
of Pennsylvania
March 5, 1979; revised May 30, 1979
Partially purified cytochrome d free of other cytochrome components has been isolated using detergent (Triton X-100) and salt (KCl). The isolated cytochrome d, has an absorption maximum at 615-617 nm and is capable of oxidizing the reduced cytochrome cjsa of tetrahymena. Potentiometric titrations show that cytochrome d like cytochrome aa has a twocomponent structure with half-reduction potentials of 0.17 and 0.32 V. EPR studies of aerobic cytochrome d show low spin ferric heme resonances at g = 2.96 and g = 2.26 and a g = 2.0 signal characteristic of oxidized copper. EPR studies on oriented multilayers of partially purified cytochrome d show that the heme seen in the fully oxidized preparation is oriented in such a way that the angle between the heme normal and the membrane normal is approximately 90”.
It has been known since the early studies versity of Pennsylvania) was grown and harvested as of Warburg and his co-workers (1, 2) and described previously (6) and mitochondria were isoKeilin (3) that pigments other than cyto- lated according to the method given in Ref. (6). The chrome uu3 can function as the terminal oxi- final mitochondrial pellet was suspended at a protein concentration of approximately 40 mg/ml in 0.25 M dases in bacterial systems. One of these al- sucrose-10 mM KCl-5 mM Mops*-0.2 mM EDTA conternate oxidases, cytochrome d (a,), was taining 0.2% bovine serum albumin and 10% dimethyldiscovered by Yaoi and Tamiya (4) in Esche- sulfoxide (pH 7.4), quickly frozen in liquid nitrogen, richia coli and Shigella dysenteriae and fur- and stored below -18°C. ther investigated by Keilin (5) in Axotobacter. We have shown previously (6) that cyto- Zsolation and Partial PuriIcation of chrome d is the terminal oxidase in the ciliCytochrome d ate protozoan, Tetrahymena pytiformis. In The mitochondrial suspension was thawed, diluted this paper we describe an isolation tech- approximately 15-fold in 0.25 M sucrose-O.01 M phosnique which yields a partially purified active phate buffer (pH 7.2), and centrifuged at 10,000 rpm cytochrome d. The properties of cyto- for 10 min. The pellet was resuspended in 0.25 M suchrome d as well as its orientation with re- crose-0.01 M phosphate buffer (pH 7.2) at a protein spect to the plane of the membrane are dis- concentration of 30 mgiml and treated with 10% Triton cussed and compared to those of cytochrome X-100 (final concn = 0.7 mg/mg protein) and solid KC1 (final concn = 1 M). The pH was maintained at 7.4 by addition of 0.5% ammonia in water. The mixture was stirred for 20 min at 4°C and centrifuged for 45 min at 32,000 rpm in a Beckman Model L5-50 ultracentrifuge. After centrifugation, several distinct fractions were obtained. The top, light pink supernatant layer
aa3. METHODS AND MATERIALS Z’etrahymena pytiformis, strain ST, (kindly provided by Dr. Y. Suyama, Department of Biology, Uni-
(about 90% of the total supernatant)
contained soluble
mitochondrial proteins and the remaining cytochrome
’ To whom correspondence should be directed. Supported by USPHS HL 18-708 and GM 12202. M.E. is an Established Investigator of the American Heart Association.
2 Abbreviations used: Mops, 3-(N-Morpholino)propanesulfonic acid. 1
WO3-986~79/11099&09$02.00/0 Copyright 0 1979 by AcademicPress, Inc. AU rights of reprodn&ion in any form reserved.
2
KILPATRICK
AND ERECINSKA
c and b,, pigment. It was pipetted off and discarded. The remaining bottom, supernatant layer was dark red and appeared quite viscous. It contained the bulk of the cytochrome be, complex. The pellet consisted of a tightly packed black sediment surrounded by a loosely packed greenish-brown layer which contained cytochromed. Since it was difficult to separate the be, complex from the oxidase layer at this stage, the dark red supernatant layer and the bottom greenish-brown sediment were suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) and centrifuged for 30 min at 32,000 rpm. The tightly packed black pellet was discarded. Good separation was achieved; the cytochrome d formed a tight greenish brown pellet whereas the bc, complex remained soluble in the supernatant. The supernatant was discarded and cytochrome d was resuspended in a minimal amount of 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2).
Analytical
Methods
Absolute and dvference spectra were obtained using a Johnson Foundation dual wavelength scanning spectrophotometer adapted for use with a PDP-11 computer. The detailed conditions are given in the figure legends. Cytochrome d activity. The activity of partially purified cytochrome d was determined by measuring the rate of oxidation of isolated reduced T. pyriformis cytochrome c5= by the solubilized cytochrome d fraction. Isolated cytochrome es53(7) was reduced with sodium dithionite and filtered through a Sephadex G-25 column to remove excess dithionite. The rates of oxidation were measured in a dual wavelength spectrophotometer by monitoring the decrease in absorption at 553540 nm at 22°C. Cytochrome d activity was also measured in the presence of two inhibitors, potassium cyanide and sodium sulfide. The experimental conditions were the same as described above (see figure legends for the details). Determination of copper and heme contents. The copper content of isolated cytochrome d was determined by two different methods. In the first one, copper was measured calorimetrically by the biquinoline method of Brumby and Massey (8) using cupric sulfate as the standard. In the second, the oxidized copper signal at g = 2.0 was quantitated by double integration using a Varian E-109 spectrometer equipped with a Model 1074 instrument computer (Nicolet Instt-ument Corporation) and compared with double integrated signals of copper-EDTA standards at concentrations between 10 pM and 1 mM. All measurements were made using nonsaturating microwave powers at 18 K. An approximation of the heme content was made by comparison of the low spin ferric heme resonance of cytochrome d at g = 2.96 with the low spin ferric heme resonance at g = 3.0 of mammalian cytochrome aa prepared from pigeon breast mitochondria.
Protein Determination. Proteins were assayed by the biuret method (9) using crystalline albumin (Sigma Chemical Co.) as a standard. Potentiometric titrations were carried out by simultaneous measurements of absorbance (dual wavelength spectrophotometer) and the oxidation-reduction potentials (redoxostat) at 22°C. The redoxostat used is similar to that described by Swartz and Wilson (10) with modifications by Wilsonet al. (11). The redoxostat utilizes four electrodes; the measuring electrodes are glassy carbon with a standard calomel reference, the working electrode is glassy carbon with Ag-AgCl counter electrode. Methyl viologen (Sigma Chemical Co.) was used as the primary electron acceptor for the working electrode, while ferrocenyl methylitrimethyl ammonium iodide (ICN Pharmaceuticals, Inc.) and ferricyanide acted as electron donors. The oxidase (approx. 15-20 mg protein/ml) was suspended in 0.25 M sucrose-O.01 M Pi, pH 7.2 and phenazine methosulfate (Sigma Chemical Co.) and diaminodurene (Aldrich Chemical Co.) were used as redox mediators. The results are presented graphically as the logarithm of the ratio of oxidized to reduced forms of the cytochrome (abscissa) as a function of the oxidationreduction potential (ordinate). The titration curves were separated into individual components as described by Wilson and Dutton (12)and Duttonet al. (13). Electron
paramagnetic
resonance
measurements.
EPR spectra were measured using a Varian E-109 spectrometer equipped with an Air Products LTD 3-110 liquid helium cryostat. Preparation oforiented cytochrome d. Oriented multilayers of tetrahymena cytochrome d were prepared as deseribed by Erecinska et al. (14). It was found that thin samples (-11 mg protein for a l-cm-diameter specimen) required less time for partial dehydration and produced better oriented specimens.
RESULTS
Isolation
of Cytochrome
d
The procedure described above provides a method for isolating cytochrome d of T. pytiformis. Although the method appears simple, the main problem lies in that cytochrome d is highly unstable and denatures easily (15). Therefore standard methods used for isolating cytochrome aa which utilize extensive fractionation in the presence of salt and detergent cannot be applied to isolate an active cytochrome d. In developing the isolation procedures we have found that certain precautions must be taken with the storage of the tetrahymena mitochondria to insure isolation of a spectrally un-
ISOLATION
AND CHARACTERIZATION
modified enzyme. The mitochondria must be rapidly frozen in liquid nitrogen in the presence of dimethyl sulfoxide to prevent formation of ice crystals which disrupt the mitochondrial structure. Moreover, they should be stored either in liquid nitrogen or at below -20°C in a freezer which maintains a constant low temperature and does not have a defrost cycle. Better yields of cytochrome d were consistently obtained from mitochondria which were stored for less than a week. The first centrifugation after thawing of the mitochondria can usually provide an indication as to the yield and quality of cytochrome d. If the supernatant is pink due to the liberation of the loosely bound cytochrome es53it has been our experience that subsequent cytochrome d yield will be very small and the enzyme will not be active. When the precautions described above were taken, the procedure yielded in our hands reproducible and reliable results. An overall yield of 84% was obtained by calculating the recovery of heme d in the oxidase fraction from a known concentration of mitochondria. The purification was threefold as measured by the increase of heme d content per milligram protein. This
OF CYTOCHROME
d
3
low purification value is due to the fact that the partially purified cytochrome d, although deficient in other cytochromes, contains nevertheless relatively high amounts of colorless proteins. Attempts of further purification were unsuccessful because any additional manipulation led to modification and inactivation of this enzyme. Characteristics
Cytochrome
of the Isolated d
Figure 1 (I) shows the spectral properties of the isolated cytochrome d in the visible region. The sharp asymmetric peak at 61’7 nm is due to cytochrome d whereas a small peak at 560 nm may be attributed to contamination with the cytochrome b-c 1 complex (less than 10%). The absorption spectra of the isolated oxidized and reduced cytochrome d are presented in Fig. 1 part II, A and B, respectively. The Soret absorption maximum of oxidized cytochrome d is at 420 nm while the reduced form exhibits a Soret peak at 445 nm. The difference spectrum for the isolated cytochrome d in the Soret region (reduced-oxidized) is shown in Fig. 1, part
FIG. 1. The absolute and differenee spectra of isolated cytochrome d in the (Yand Soret region. The absolute and difference spectra were obtained using a PDP-11 computer operated scanning dual wavelength spectrophotometer. (I) The difference spectrum of isolated cytochrome d in the a and p region. Cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer at pH 7.2 at a protein concentration of 0.29 mg/ml. The spectrum of the fully oxidized sample was measured and stored in the computer memory as the baseline. The sample was reduced with dithionite and measured as (dithionite-reduced) - oxidized. The reference .wavelength was 575 nm. (II) The absolute spectra of isolated cytochrome d in the Soret region. Cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 0.14 mgimi. The reference wavelength was 460 nm. The spectrum of the buffers alone were measured and stored in the eomputer memory as the baseline. (A) Fully oxidized - buffer. (B) (Dithionite-reduced) - buffer. (C) (Dithionite-reduced + CO) - buffer. (III) The difference spectra of isolated cytochrome d in the Soret region obtained from the absolute spectra shown in part II. (A) (Dithionite-reduced)-oxidized. (B) (Dithionite-reduced + CO)-oxidized.
4
KILPATRICK
AND ERECINSKA
III, trace A and has an absorption maximum at 444 nm.
dized and reduced cytochrome d in the Soret region of the spectrum. Although the spectral changes are small, they are clear enough to conclude that cyanide binds to both the reduced and the oxidized forms of the enzyme.
Ligand Binding The effects of ligand binding on the absorption spectra of isolated cytochrome d were studied using the following ligands: carbon monoxide, nitric oxide, and cyanide. In Fig. 1 (parts II and III) the absolute and difference spectra of the CO-cytochrome d complex are shown in the Soret region. The absolute spectrum of the COcompound was obtained by bubbling the dithionite-reduced sample with CO gas for a period of 3 min. It can be seen that the CO-difference spectrum [(dithionite reduced + CO) - (dithionite reduced)] of cytochrome d exhibits an absorption maximum at around 428 nm. Figure 2, part I (trace C) shows the absolute spectrum of the NO-cytochrome d complex in the Soret region obtained upon the addition of sodium nitrite to the dithionite-reduced sample. A broad peak is observed with an absorption maximum at around 430 nm. Figure 2, parts II and III, shows the effect of cyanide addition to oxi-
Copper and Heme Determinations Previous preliminary EPR studies (‘7) suggested that cytochrome d, like cytochrome aa3, contains copper. Measurements of the copper content calorimetrically on partially purified preparations showed it to be 1.48 2 0.01 nmol copperlmg protein. The heme content of the preparation was calculated making the assumption that the extinction coefficient for cytochrome d at 617-640 nm was 25 mM-’ cm-‘. [It should be mentioned that attempts were made to convert heme d to its alkaline hemochromogen (6) and thereby determine its concentration. However, this led to modification of the heme into a compound for which the spectral properties are not known.] Calculation of the heme content using the above extinction coefficient gave a value of 0.85 4 0.11 nmol heme/mg protein, i.e., -0.5 m
400
420
440
460 Alnm)
480
500
400
420
440 hlnml
460
480 -a
400
440
480
h(nm)
FIG. 2. The effects of cyanide and NO addition on the absolute spectra of isolated cytochrome d in the Soret region. The absolute spectra were obtained using a PDP-11 computer-operated scanning dual wavelength spectrophotometer with a reference wavelength at 460 nm. Isolated cytochrome d was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 0.14 mg/ml. The spectrum of the buffer alone was measured and stored in the computer memory as the baseline. (I) (A) Fully oxidized sample - buffer. (B) (Dithionite-reduced sample) - buffer. (C) (Dithionite-reduced sample + NO) - buffer. The sample was reduced with a few crystals of sodium dithionite and 50 mM sodium nitrite was added. (II) (A) Fully oxidized sample - buffer. (B) (Fully oxidized sample + 5 mM KCN) - buffer. (C) (Dithionite-reduced sample with KCN present from B) - buffer., (III) (A) Fully oxidized sample - buffer. (B) (Dithionite-reduced sample) - buffer. (C) (Dithionite-reduced sample + 1 mM KCN) - buffer.
ISOLATION
AND CHARACTERIZATION
OF CYTOCHROME d
5
heme to 1 copper. However, if cytochrome d contains two hemes [as shown in the po-
tentiometric titrations in Ref. (6) and Fig. 31 and if by analogy with cytochrome uu3 only one of the hemes is responsible for the absorption properties in the visible region, then the heme to copper ratio would be close to unity. These findings were substantiated by results obtained with EPR spectroscopy. Double integration of the copper signal in the oxidized preparation of cytochrome d yielded a value of approximately 0.7 nmol of copperlmg protein which is about one-half the value obtained calorimetrically. This suggests that only one of the two coppers of cytochrome d is detectable by EPR measurements. The heme content of isolated cytochrome d was estimated by comparing the low spin ferric heme resonance at g = 2.96 with the g = 3.0 signal of isolated cytochrome ua3 whose heme content has been calculated from optical absorbance measurements [millimolar extinction coefficient at 605 nm (reduced) - 630 nm (oxidized) = 26.4 cm-‘]. Such estimates gave a value of approximately 0.6 nmol of cytochrome d heme/mg protein. Therefore, the EPR measurements yielded a heme to copper ratio of 1 to 1. Since only one of the coppers of cytochrome d is detectable by EPR, it is reasonable to conclude that only one of the hemes is detectable. This is similar to the situation seen with mammalian oxidase. Activity
of Zsolated Cytochrome
d
In order to establish that isolated cytochrome d is still functional, its ability to oxidize reduced tetrahymena cytochrome c5% was measured and compared with values obtained with tetrahymena oxidase in intact mitochondria (7). Isolated cytochrome d was able to oxidize reduced tetrahymena cytochrome es53at rates comparable to those seen in intact mitochondria and as shown in Fig. 3 part I, the rate of oxidation increased -proportionally with increasing concentrations of cytochrome d. By extrapolation from the initial velocity rates of oxidation of reduced cytochrome css3,double-reciprocal plots were obtained which gave a K, ‘of approximately 20 ~.LM
FIG. 3. Activity of isolated cytochrome d. Part (A). Reduced tetrahymena cytochrome css3(24 pM) was suspended in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at room temperature. The reaction was started by the addition of various concentrations of isolated cytochrome d (A: 0.24 mg protein, B: 0.48 mg protein, and C: 0.96 mg protein). The rate of oxidation was measured spectrophotometrically at 553-540 nm. (Part B). The effects of cyanide and sulfide on the activity of cytochromed. The conditions are the same as described above using 24 pM cytochrome cAa3and 0.96 mg protein cytochrome d.
(not shown). This value is similar to the K, of 5-10 &&M obtained with intact mito-
chondria using the same procedure. To further establish that cytochrome d has not undergone modification during isolation, the effect of inhibitors on the rate of oxidation was measured. Part II of Fig. 3 shows that the addition of cyanide and sulfide completely inhibits oxidation of reduced cytochrome es53by cytochrome d. In intact mitochondria using NADH as substrate, it was found that 1 mM KCN inhibits oxidation of this substrate by 90%, 50 mM Na,S by 85%, 50 mM hydroxylamine by 65%, and 50 mM azide by 40% (data not shown). Therefore, it appears that cytochrome d isolated by this method has not undergone any severe modification. Furthermore, it shows
6
KILPATRICK
AND ERECINSKA
the same response to inhibitors as the membrane-bound enzyme. Oxidation-Reduction Potentials Isolated Cytochrome d
of
-
/-Lfr ,-?9h
I
’ h------
Anaerobic potentiometric titrations of suspensions of isolated cytochrome d were carried out as described in methods and the results are shown in Fig. 4. The titration FIG. 5. The EPR spectra of partially purified yields a sigmoidal curve which can be re- aerobic cytochrome d. Cytochrome d was suspended solved into two components with n = 1 and in 0.25 M sucrose-O.01 M phosphate buffer (pH 7.2) at half-reduction potentials of 0.170 and 0.320 a protein concentration of 22 mgiml and frozen in liquid V at pH 7.2 and 22°C. These midpoint nitrogen in quartz EPR tubes. EPR frequency of 9.106 potential values are close to those obtained GHz, microwave power 5 mW, and temperature 15°K. from titrations of cytochrome d in intact Scanning time: 4 min, time constant: 1s. tetrahymena mitochondria (6) and submitochondrial particles (7). plane of the membrane at various angles to the direction of the magnetic field are shown EPR Spectra of Isolated Cytochrome d in Fig. 6. The magnitudes of the compoThe EPR spectrum of partially purified nents of the g tensor exhibit dependence on the direction of the applied magnetic field. aerobic cytochrome d sample is shown in The lowest field signal at g = 2.94 is maxiFig. 5. The low spin ferric heme signals mal when the planes of the oriented multiat g = 2.96 and g = 2.27 are due to cytolayers are parallel to the magnetic field and chrome d. A prominent g = 2.0 signal is minimal when the planes of the membranes characteristic of oxidized copper. are normal to the magnetic field. The two maxima of the g = 2.94 signal which are EPR Spectra of Oxidized Cytochrome d in seen in the plot of signal magnitudes at variOriented Multilayers of Partially ous angles (Fig. ‘7) are exactly 180” apart. Purified Enx yme The g = 2.26 signal exhibits opposite orienThe EPR spectra of the oriented multi- tation. It is maximal when the plane of the layers of oxidized cytochrome d with the membrane is parallel to the direction of the magnetic field. The maxima are also 180” apart and 90” out of phase with the g = 2.94 resonance. DISCUSSION
The results presented in this work offer a simple and quick procedure for the preparation of cytochrome d essentially free of other cytochrome components. This is, to our best knowledge, the first successful attempt of isolation of this bacterial-type oxidase. The isolated cytochrome d has the same FIG. 4. Potentiometric titration of cytochrome d. spectral properties as does the hemeprotein Cytochrome d was titrated as described under Matein intact T. pytiformis mitochondria and rials and Methods by following absorbance changes at catalyzes the oxidation of reduced cyto617-575 nm. Cytochrome d was suspended in 0.25 M chrome cSS3by oxygen at a comparable sucrose-O.01 M phosphate buffer (pH 7.2) at a protein concentration of 15 mgiml. The redox mediators used velocity. Two components are observed during anaerobic potentiometric titrations are listed under Materials and Methods. The different of partially purified cytochrome d with halfsymbols represent oxi’dative and reductive titrations. Solid lines represent theoretical n = 1. reduction potential values slightly lower
ISOLATION
AND CHARACTERIZATION
OF CYTOCHROME d
Tetrahymeno Pyrlformls Oxldase
nrg=226
3
40”
2000
II
2200
/
2400
2600
2800
300
Magnetic Field (Gauss1
MAC-498
FIG. 6. EPR spectra of oxidized cytochrome d in oriented multilayers of the “membranous” enzyme at various angles between the direction of the magnetic field and the normal to the membrane plane. The oriented l-cm multilayer was formed from 11 mg protein of cytochrome d and partially dehydrated for approximately 48 h at 90% relative humidity and 4°C. The partially hydrated multilayer with its supporting mylar was cut into 2-mm strips and inserted into a quartz capillary tube and frozen in liquid nitrogen. EPR frequency: 9.11 GHz, microwave power: 10 mW, sample temperature 6.5”K, scanning time: 2 min, time constant: 0.25 s, and modulation amplitude: 2.5 x 10 G;
than those obtained for the native system (0.32 vs 0.345 V and 0.17 vs 0.245 V). This shift in the half-reduction potential values may be caused by the presence of a detergent which modifies the environment of the heme. Such changes have been observed in other preparations of mitochondrial complexes in the presence of detergents (16). The two-component nature of cytochrome d may be, like that of cytochrome aa3, the intrinsic property of this enzyme. Cytochrome d is capable of binding carbon monoxide and forming a characteristic reduced cytochrome d-CO complex. The EPR studies on isolated cytochrome d showed the presence of a low spin ferric heme which exhibits resonances at g = 2.94-2.96 and g = 2.26. The magnitude of the highest field g signal was too small to determine its position with sufficient ac-
Tefrahymena Pyriformls Oxldase g’226
60? 7.4 50E 3
,_
l=“’
\y , , I
r, \
:
0 20”
60’
100” Angle
140”
180’
220”
MM - 49,
FIG. 7. Plot of relative EPR signal intensities for oxidized cytochrome d in oriented multilayers as a function of the angle between the magnetic field direction and normal to the plane of the membrane. The EPR spectra were recorded under conditions specified in Fig. 6.
8
KILPATRICK
AND ERECINSKA
curacy. In addition, the presence of a pronounced g = 2.0 signal characteristic of oxidized copper, suggests that cytochrome d, like cytochrome aa3 is a heme-copper protein. This conclusion is supported by chemical analysis which shows copper to be present in the preparation in amounts comparable to those of the heme( Successful preparation of hydrated oriented multilayers from partially purified cytochrome d permitted the determination of the orientation of the heme chromophore(s) with respect to the plane of the multilayers. Since the heme structure of cytochrome d as well as the three-dimensional configurations of the enzyme are not known at the present time, the interpretation which we offer involves certain assumptions and relies on the information obtained from single crystal analysis of cytochrome c (17) and low spin ferric derivatives of myoglobin (18,19). The lowest field g resonance at g = 2.942.96 is maximal when the planes of the multilayers are parallel to the magnetic field (Figs. 6 and ‘7). Thus, if one assumes that this resonance arises from the g, component of the g tensor (i.e., the one which is approximately normal to the plane of the heme (20)) the conclusion can be drawn that the oxidized heme of cytochrome d is oriented normal to the plane of the multilayer. This also means that the oxidized heme of cytochrome d is similarly oriented to that of cvtochrome aan. Such an orientation was measured for the latter heme in oriented multilayers from a “membranous” form of the isolated enzyme (21, 22) in oriented multilayers of mitochondrial membranes (23), and their fragments (24), and in oriented multilayers from a bacterium P. denitrificans
(25).
The g = 2.26 resonance is maximal when the planes of the membranes are normal to the magnetic field. Assuming that this resonance arises from the gl, component of the g tensor (i.e., one of the two principal components of the g tensor which lie in the plane of the heme (20)) one can conclude that the g, axis is oriented parallel to the planes of the membranes. This orientation of the gU axis in fully oxidized cytochrome d is different from that of cytochrome aa3 from pigeon breast mitochondria and P. denitrif-
icans (25). In view of the very limited knowledge of the chemistry of cytochrome d it is difficult to draw any further conclusion from this finding. In summary, cytochrome d has been partially purified from mitochondria of T. pyriforrnis and its spectral and thermodynamic properties have been measured. The orientation of the heme of cytochrome d with respect to the planes of the oriented multilayers has been determined. The availability of yet another terminal oxidase may prove helpful in our understanding of the operation of cytochrome oxidases. REFERENCES 1. WARBURG, O., AND NEGELEIN, E. (1929) Biothem. Z. 214, 64-100. 2. WARBURG, O., NEGELEIN, E., AND HAAS, E. (1933) Biochem. Z. 266, 1-8. 3. KEILIN, D. (1927) C. R. Sot. Biol. Paris 96, sp39-Sp68. 4. YAOI, H., AND TAMIYA, H. (1928) Proc. Imp. Acad. Japan 4, 436-439. 5. KEILIN, D. (1933) Nature (London) 132, 783. 6. KILPATRICK, L., AND ERECI~~SKA, M. (1977) Biochim. Biophys. Acta 460, 346-363. 7. KILPATRICK, L., AND ERECI~SKA, M. (1977) Biochim. Biophys. Acta 462, 515-530. 8. BRUMBY, P. E., AND MASSEY, V. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 463-474, Academic Press, New York. 9. GORNALL, A. B., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 10. SWARTZ, D. B., AND WILSON, G. S. (1971) Anal. Biochem. 40, 392-400. 11. WILSON, D. F., ERECI~~SKA, M., SANDLER, S., AND NELSON, D. (1979) in International Symposium on Oxidases and Related OxidationReduction Systems, III (King, T. E., Mason, H., and Morrison, M., eds.), Albany, N.Y., in press. 12. WILSON, D. F., AND DUTTON, P. L. (1970) Biothem. Biophys. Res. Commun. 39, 59-64. 13. DUTTON, P. L., WILSON, D. F., AND LEE, C. P. (1970) Biochemistry 9, 5077-5082. 14. ERECI~~SKA, M., BLASIE, J. K., AND WILSON, D:F. (1977) FEBS Lett. 76, 235-239. 15. LLOYD, D., AND CHANCE, B. (1972) Biochem. J. 128, 1171-1182. 16. LEIGH, JR., J. S., AND ERECI~~SKA, M. (1975) Biochim. Biophys. Acta 387, 95-106. 17. MAILER, C., AND TAYLOR, C. P. S. (1972) Canad. J. Biochem. 50, 1048-1055. 18. HELCKE, G. A., INGRAM, D. J. E., AND SLADE, E. F. (1968) Proc. Roy. Sot. B 169, 275-288.
ISOLATION AND CHARACTERIZATION OF CYTOCHROME d 19. H0~1,H.(1971)Biochim.Biophys. Acta251,227235.
9
23. ERECI~~SKA, M., WILSON,D. F., AND BLASIE, J. K. (1978)Biochim.Biophys. Acta 501,63-‘72.
20. GIBSON,J.F., ANDINGRAM,D.J. E. (1957)Na24. BLUM, H., HARMON, H. J., LEIGH, J. S., ture (London) 180,29. SALERNO,J. C., ANDCHANCE,B. (1978)Bio20. BLASIE,J.K.,ERECII;JSKA,M.,SAMUELS,S.,AND chim. Biophys. Acta 502, l-10. LEIGH,JR., J. S. (1978)Biochim. Biophys. Acta 501, 33-52. 25. ERECI~SKA,M., WILSON,D. F., AND BLASIE, 22. ERECI~~SKA, M., WILSON,D. F., AND BLASIE, J. K. (1979)Biochim. Biophys. Acta 545, 352J. K. (1978)Biochim. Biophys. Acta 501,53-62. 367.
