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Spectrophotometric studies of the pigments of adrenal cortex mitochondria
ARCHIVES
OF
BIOCHEMISTRY
AND
Spectrophotometric
BIOPHYSICS
122, 735-747 (1967)
Studies Cortex
WENDY Departtrlent
CAMMER2
of the
Pigments
of Adrenal
Mitochondria’ RONALD
AND
of Biophysics rind Physical Cnioer,sity of Pennsylvania, lteceived
Biochemistry, Philadelphia, August
W. ESTABROOKS Johnson Research Foundation, Pennsylvania 19104
8, 1967
St,rldies have been carried out to determine the spectral changes occurring during srlccinate and malate oxidation by mitochondria from beef adrenal cortex. The conThe spectral tent of cytochromes a + a3 , b, c + cl , and P-450 have been determined. propert.ies of redllced cgtochrome P-450, its complex with CO, and the interaction of oxidized cytochrome P-450 with deoxycorticosterorle have been determined. as well as the location of absorption The changes in extinction coefficients, maxima and minima occrlrring dluing cptochronle P-450’s oxidation arid redrlctioll associated with hydroxylatiorr of LNC, are described. The results obtained are conpared with those for cytochrome P-450 of microsomes prepared from rabbit liver.
adrenal cortex mitochondria (2, 12) and has been implicated in the lip-hydroxylase rea&on (13-16) as well as in t’he conversion of cholesterol to pregnenolone (4, 17). Spectral studies of reduced and oxidized cytochrome P-450 in microsomes are complicated by interference from cytochrome bg (18), and attempts to isolate cytochrome I’-450-containing fractions from mi~rosomal membranes have led to obvious alterations in its spectral propertries (19-21). Recently a cytochrome I’-450-enriched fraction has been obtained (14) from adrenal cortex mitochondria, and permitt’ed the measurement (14, 22, 23) of some of the spectral properties of cytochrome P-450. To assess the possible alterations occurring to cytochrome l’-450 during its isolation, the spect)ral studies of adrenal cortex mitochondria reported here were undertaken. In addition t’he present study examines the changes of cytochrome l’-430 occurring during steroid hydroxylation, and confirms (2) the presence of a normal mitochondrial respiratory chain in adrenal cort,ex mitochondria. These studies also support the increasing body of evidence for an interact’ion in adrenal cortex mitochondria between a normal respiratory chain
Nit,ochondria isolated from the cortex of the beef adrenal gland carry out the reactions of oxidative phosphorylation characteristic of mitochondria isolated from heart,, liver, kidney, etc. (1). In addition these mtochondria also carry out certain steroid hydroxylation reactions (2-6), most not’ably the 1I@-hydroxylation of deoxycorticosterone (7, 8). The lip-hydroxylase reaction of adrenal cortex mitochondria has been shown to resemble ot,her mixed function oxidations in incorporatjing 180 from molecular oxygen (9) into the subst’rate molecule (deoxycorticosterone, DOC) and in t,he stoichiometry for oxygen and DOG (1). Furthermore, cyt’ochrome P-450, which is functional in many microsomal mixed function oxidations (10, II), has been shown to be present, in 1 These stIltlies were supported in part by Granls from the U.S. Public Health Service ((+M 12202) and the National Science Folmdatioll (GE 2451) . 2 Predoctoral Fellow of t,he GradLlate Group on Molewdar Biology; slIpported by U.S. Public IIealth Service grant 5 TO1 GM-00694.07. 3 These stlldies were carried ollt, drlrilrg the tenrlre of a U.S. Prlblic Health Service Research Career Development Award (GM-K3-4111). 735
736
CAMMER
AND
containing cytochromes a, u3, b, and c + cl and a second type of respiratory chain leading to a steroid hydroxylation and containing cytochrome P-450 (2, 3, 15, 16, 24). METHODS The preparation of beef adrenal cortex mitochondria has been described previously (1). For the experiments reported here, the isotonic bluffer employed contained 0.25 M sucrose, 20 mM KCl, 10 mM potassium phosphate buffer, pH 7.4, 15 mM triethanolamine chloride buffer, pH 7.4, and 5 rnM MgC12. Sodium succinate and sodium malate were prepared as 1 M solutions which had been neutralized to pH 7. Deoxycorticosterone (DOC) was obtained from the Sigma Chemical Company, St,. Louis, Missouri, and was prepared as a 48 mM solution in ethanol. The wavelength scanning recording spectrophotometer has been described by Chance et al. (25). Protein determinations were carried out by the method of Gornall et al. (26). RESULTS
Cytochromes of adrenal cortex mitochondria. It has been established that Krebs’ tricarboxylic acid cycle substrates are capable of supporting the lip-hydroxylation of DOC by reducing the endogenous mitochondrial pyridine nucleotides (2,3,27-29) and thence cytochrome P-450, via a flavoprotein and a nonheme iron protein (30, 31). When succinate is used as substrate, reduction of these electron carriers, including cytochrome P-450, is prevented by inhibitors of the normal mitochondrial respiratory chain. Accordingly it was considered possible to examine spectrophotometrically the pigments reduced when succinate was used as substrate in order to reduce cytochrome P-450 as well as the normal respiratory chain cytochromes of adrenal cortex mitochondria, and to compare this result with spectra obtained when succinate was added in the presence of cyanide, where only the cytochromes of the normal respiratory chain and not cytochrome P-450 were reduced, i.e., inhibition of the normal respiratory chain prevents the formation of malate from succinate or the generation of energy required for the succinate-linked reduction of pyridine nucleotides, and thereby prevents reduction of cytochrome P-450. In order to obtain the difference spectra
ESTABROOK
shown in Fig. 1, a suspension of adrenal cortex mitochondria was diluted in isotonic buffer and divided equally between two cuvettes, and the spectra were recorded with a wavelength scanning recording spectrophdtometer. Trace A was obtained after the addition of cyanide and succinate to the experimental cuvette. The absorption bands with maxima at 605 and 445 rnp, 561 and 428 rnp, and 552 and 419 rnp are characteristic of the normal respiratory chain cytochromes (a + u3), b, and (c + cJ, respectively. From spectral studies of this type the concentrations of normal respiratory chain cytochromes in adrenal cortex mitochondria were determined, and the results are summarized in Table I. The values obtained with adrenal cortex mitochondria agree quite well with the cytochrome content of liver mitochondria (30). In contrast, the cytochrome content of beef adrenal cortex mitochondria reported by Harding and Nelson (2) are considerably higher than those found here. In the experiments of Harding and Nelson, this discrephncy can be attributed to interference from the spectral contributions of cytochrome P-450 (see below). The contribution of cytochrome P-450 leads to deceptively high values for cytochromes b and c, as illustrated by the difference spectrum (curve B) presented in Fig. 1. In this experiment an aliquot of beef adrenal cortex mitochondria was diluted as described above, and succinate was added as substrate in the absence of cyanide. After a brief period of incubation, an increase in absorbancy at 445 rnp indicated reduction of cytochrome a3 associated with exhaustion of oxygen in the reaction cuvette. The spectrum obtained under these conditions is similar to that reported by Harding and Nelson (2). Spectrum of cytochrome P-450. In order to determine the contribution of those pigments reduced by succinate in the absence of cyanide relative to those pigments reduced by succinate in the presence of cyanide, the difference spectrum presented in Curve C of Fig. 1 was recorded. This spectrum represents the difference spectrum of reduced cytochrome P-450 minus oxidized cytochrome P-450. A broad a-band with a
WECTRA
OF ADRENAL
CORTEX
MITOCI-IONDRIA
428 /
28 ,445
Succinote+ KCN Minus Oxidized B --- Succinate(onoerobic1 Minus Oxidized C..... Succinote(onoerobic) Minus Succinote+ KCN
A-
I 1 Wovelength
I
I
I
, I 550
I
I,,
I 600
I
I
I
, I 650
FIG. 1. Spectral changes of adrenal cortex mitochondria occurring during cytochrome reduction with succinate as substrate. Adrenal cortex mitochondria were diluted to 6 ml with bluffer mixture and divided equally into two cuvettes. Succinate (5 mM) and potassium cyanide (1 rnw) were added to one cuvette, and the difference spectrum shown in Curve A (solid line) was recorded. An equivalent dilution of mitochondria was divided equally in two cuvettes, and succinate (5 mM) was added in the absence of cyanide. After utilization of the oxygen dissolved in the reaction medium had occurred, Curve B (dashed line) was recorded. Succinate (5 mM) and potassillm cyanide (1 mM) were then added to the other cuvette (referenre cuvette), and the difference spectrum shown in Curve C (dotted line) was recorded. The visible spectra were obtained at a protein concentration of 3.7 mg/ml, while the Soret spectra were recorded with a protein concentration of 1.7 mg/ml.
maximum at about 555 mp and a Soret band with a maximum at 432 rnp are observed. In beef adrenal cortex mitochondria the spectral contributions of cytochrome P-450 are significantly greater than those of cytochrome b and c + cl of the normal respiratory chain. The positions of the maxima for the spectral contribution of reduced minus oxidized
cytochrome
P-450 agree well
with those found by Cooper et al. (18) using adrenal cortex microsomes. While recording these spectra, it was noted that there frequently was a significant decrease in absorbance not attributable to a change in the oxidation reduction of a cytochrome pigment. This is illustrated by the negative displacement from the baseline in
Fig. 1, curves B and C. This decrease in absorbance could be caused either by a swelling of mitochondria in the presence of substrate or to the spectral changes associated with reduction of the flavoprotein and the nonheme iron protein functional in the electron transport system from TPNH to cytochrome P-450. The large decrease in absorbance at about 460 m,u, as well as the relatively high content of nonheme iron protein as determined by electron paramagnetic spectroscopy (Table I), suggest that the wavelength independent loss of absorbance in the visible region of the spectrum is possibly due to reduction of flavoprotein and nonheme iron protein. Previous studies (1, 12, 28) have shown
738
CAMMER
AND
ESTABROOK
TABLE CONTENT
OF
RESPIRaTORY
ClRRIERS
I
OF
ADRENAL
CORTEX
MITOCHONDRL~” Content
Pigment
Cytochrome a + a3 b c -t Cl P-450 Nonheme iron TPN + TPNH DPN + DPNH
Method
Spectrophotometric
Spectrophotometric wavelengths 6OFG-625mp 562-575 mp 551-540 mu 45C490 rnfi EHR spectrometry Enzymic assay Enzymic assay
(mab : cm-l)
16 20 19 91
Ad&al mitochondria
0.23 0.17 0.29 1.5 2.6 2.8 6.3
(mjmoles/mg
protein)
Adrenal mitochondria
Liver mitochondria
0.756 0.32’ 0.67’ 1.35
0.22( O.lW O.Xlc
2.gd 5.5d
a Respiratory chain cytochromes were determined from difference spectra of the reduced minus oxidized pigments using the extinction coefficients indicated (32). Cytochrome P-450 was determined from the magnitude of the absorption band of the reduced cytochrome P-450 carbon monoxide complex using the extinction coefficient determined by Omura and Sato (19). Pyridine nucleotides were determined by enzymic assay (33), and non-heme iron was determined by comparing the magnitude of the g = 1.94 electron paramagnetic resonance signal in dithionite treated mitochondria with the signal obtained from a known amount of non-heme iron protein purified from adrenal cortex mitochondria (34, 35). * Data from Harding and Nelson (2). c Data from Estabrook and Holowinsky (32). d Data from Klingenberg (36).
that malate, as well as succinate, is a suitable substrate for supporting the maximal rate of DOC hydroxylation by adrenal cortex mitochondria. Hydroxylation occurring in the presesence of malate is relatively insensitive to cyanide when compared wit’h succinate-supported hydroxylation. When spectra were recorded with malate as substrate, t,he difference spectra presented in Fig. 2 were obtained. In the aerobic steady state (curve A) a part’ial reduction of cytochromes was observed. The reduction of flavoprotein and nonheme iron protein is ascertained by the pronounced decrease in absorbance with a minimum at about 460 rnp. After a few minutes an increase in absorbance occurred at 445 rnp, indicating reduction of cytochrome a3 and attainment of anerobiosis. The difference spectrum of malate-treated mitochondria in the anerobic state minus mitochondria in the oxidized st,ate, where malate had not been added, is shown by curve B of Fig. 2. This spectrum is similar to that shown in Fig. 1, curve B, where succinate was used as substrate. It is apparent that malate as well as succinate
are capable of reducing not only the normal respiratory chain cytochromes but also cytochrome P-450. This conclusion is supported by the spectrum presented in curve C of Fig. 2, where the malate-treated mitochondria in the anerobic state were gassed briefly with carbon monoxide. An intense absorption at about 449 rnp (note difference in scale) characteristic of the reduced cytochrome P-450 carbon monoxide complex was observed. In agreement with earlier studies of Harding and Nelson (a), t’he content of cytochrome P-450 (cf. Table I) is sufficiently high to obscure the absorbancy contributions of the other cytochromes present in adrenal cortex mitochondria. The presence of CO interaction with cytochrome oxidase is indicated, however, by the small decrease in absorbancy at 604 rnp concomitant wit,h the appearance of a shoulder at about 595 rnp. Note also the increase in absorbance of the broad complex absorption band at about 552 rnp. DOC-Induced spectral changes. Further studies were carried out attempting to establish the changes in the oxidation state of
SPECTRA A,B
OF ADRENAL
CORTEX
739
MITOCHONDRIA
C
0.16+0.2
0.08--0.1
(oeroblc)-
B. Molote (onaerobic)-Oxidized
-0.24---0.3
1
350
Oxidized
Minus Oxidized
, I
I,,
I
400
I
I
I,
I
I
I
I,
450
I
500 Wavelength
-0.06
I
I
I
,
550
I
I
I
I,
I
I
I
600
(mp)
FIG. 2. Spectral changes of adrenal cortex mitochondria associated with malate oxidation. Bs in Fig. 1, an aliquot of adrenal cortex mitochondria was diluted to 6 ml (4.5 mg protein/ml) and divided equally int,o two cuvettes. Sodium malate (5 mM) was added to one cnvette, and the difference spectrum of pigment changes during the aerobic steady state was recorded, Cllrve A (dotted line). After lttilization of oxygen in the reaction medium, the difference spectrum, Curve B (solid line), was recorded. Carbon monoxide was bubbled for about 1 minute into the contents of t,he cuvette and the difference spectrum, and Curve C (dashed line) was recorded. Note: The spect,ral changes in the region 38s490mr for Curve C were recorded with a less sensitive absorbance scale.
cytochrome P-450 without interference from absorption bands of t’he normal respiratory chain cbytochromes. In order to obtain t,he spectra presented in Fig. 3, a baseline of equal light absorbance was recorded using a sample of diluted mitochondria where the respiratory chain cytochromes were previously reduced by the addition of cyanide and succinate. Addition of the ll&hydroxylase substrate deoxycorticosterone (DOC) to the experimental cuvette resulted in decreases in absorbancy (curve A, Fig. 3) at 570 and 535 rnp, accompanying the spectral change at’ 420 rnp (14) characterist,ic of substrate interact,ion wit,h microsomal cytochrome P-4*50of these mixed funct’ion oxidase
systems reacting with steroids (37, 38) or drugs (39-41). Further addition of malate to the experimental cuvette (curve B) altered this difference spectrum only slightly, but when the llfl-hydroxylase reaction had exhausted the oxygen (1) in the experiment,al cuvett’e (curve C), one observes, as in Figs. 1 and 2, the appearance in the difference spectra of absorption bands at 553 and 433 rnp which correspond to t’he difference spectrum of the reduced form of cytochrome P-450 (cf. also Fig. 1, curve C). Of interest is the observation that the absorption band with a maximum at 432 rnp is of low absorbance relative to the baseline, which suggests the persistence anerobically of the sb-
740
CAMMER
AND
ESTABROOK
386
.06
-.06-.08
-1
575 A- - DOC minus Oxidized B ..‘... DOC Plus Malate Steady State Minus Oxidized DOC Plus Malate Anaerobic CMinus Oxidized D-- DOC Plus Malate + Na2S204 I, I I I I, I I I I
500
550
,
1 I
I
600
Wavelength (mpL)
FIG. 3. The effect of deoxycorticosterone (DOC) on pigment reduction with malate as substrate. Adrenal cortex mitochondria were diluted in buffer to 6 ml; sodium succinate (5 IIIM) and potassium cyanide (1 mu) were then added, and the suspension was divided equally into two cuvettes. The mitochondrial protein concentrations were 4.5 mg/ml for the spectra recorded from 480 to 630 rnp and 2.0 mg/ml for spectra recorded from 370 to 486 rnp. The difference spectrum shown by Curve A (dashed line) was obtained after adding DOC (240~~) to the contents of one cuvette. Sodium malate was then added, and pigments reduced aerobically during malate oxidation were recorded (Curve B, dotted line). After utilization of oxygen in the reaction cuvette, Curve C (solid line) was recorded. The subsequent addition of sodium dithionite to the anerobic sample results in a further reduction of the pigments as shown in Curve D.
sorbancy change associated with DOC addition (see below). The subsequent addition of sodium dithionite (curve D) results in further increase in absorbance at 432 mp. This further change is attributable to the loss of the DOC-induced spectral contribution with a minimum at 420 mp. To determine the contribution of DOC to the absorbance change, a similar series of experiments (Fig. 4) was carried out on a diluted mitochondrial suspension which had been treated with cyanide and succinate in order to reduce the normal respiratory chain cytochromes as described above. In agree-
ment with the results presented in Fig. 3, curve A, the addition of DOC to the experimental cuvette (trace A) showed decreases in absorbance at 570 and 421 rnp. The subsequent addition of DOC to the reference cuvette restored the baseline of equal light absorbance. The further addition of malate to the experimental cuvette (trace B) resulted in only a partial reduction of cytochrome P-450, as evidenced by the small absorption bands at 555 and 422 rnp. On exhaustion of the oxygen present in solution in the experimental cuvette (trace C), one again sees the appearance of absorption
SPECTRA
OF ADRENAL
CORTEX
MITOCHONDRIA
741
KCN Plus Succ Both A-DOC Mmus Oxldlred B.,,.... DOC Plus Malate Steady State Minus DOC CDOC Plus Malate Anaerobic
Wavelength
(mpL)
FIG. 4. The spectral changes of cytochrome P-459 during malate oxidation in the presence of DOC. Adrenal cortex mitochondria were diluted in buffer to 6 ml; sodium succinate (5 mM) and potassium cyanide (1 mM) were added, and the sample was divided equally into two cuvettes. The mitochondrial protein concentration was 5.5 mg/ml for the spectra recorded from 490 to 630 m/r and 2.4 mg/ml for spectra recorded from 370 to 490 mr. DOC (240 PM) was added to the contents of the experimental cuvette, and the difference spectrum, Curve A (dashed line), was recorded. An equal concentration of DOC was then added to the reference cuvette in order to establish a baseline of equal light absorbance. Sodium malate (5 mM) was added to the contents of the experimental cuvette, and the reduction of pigments during the aerobic steady state, Curve B (dotted line), was recorded. After utilization of the oxygen in the experimental cuvette, Curve C (solid line) was recorded. The subsequent addition of Na&04 causes a further increase in absorbance (Curve D).
bands with maxima at 555 and 432 rnp. In this instance the absorbancy change associated with the absorption band of reduced cytochrome P-450 with its maximum at 432 rnp is considerably more positive relative to the baseline than seen in Fig. 3 (curve C), and supports the conclusion that the steroidinduced trough with a minimum at 420 ml remains even in the absence of oxygen. As described above for the experiments presented in Fig. 3, the subsequent addition of sodium dithionite causes a further reduction of the cytochrome, presumably resulting
from the loss of the spectral contributions associated with DOC interaction wit.h cytochrome P-450. Effect of oxygen on Doe-induced spectral changes. Experiments were then carried out to establish directly whether the DOC-induced spectral change remains in the absence of oxygen. As shown in Fig. 5, curve A, the typical decrease in absorbance was obtained when excess DOC was added to the experimental cuvette using mitochondria diluted in isotonic buffer in the absence of any substrates or inhibitors. Malate was then
742
CAMMER
AND
ESTABROOK
d A
_--- B
_.... .. .. C
\r’
A. DOC Minus Oxidized
I I
;: i’; :I i,
B. Malate Plus DOC (anaerobic)
Minus
Malate (anaerobic)
C. Succinate
Plus DOC (anaerobic) Succinate
Minus
(anaerobic)
i..: , I
‘1,’ -420 I 1 ' ' 1 I ' ' ' ' I 1 ' ' ' , ' ' 1 1 , 400 450 500 550 60( Wavelenqth (mp)
FIG. 5. Spectral changes associated with DOC interaction with cytochrome P-450 in the aerobic and anaerobic states. A 0.6.ml aliquot of beef adrenal cortex mitochondria (45 mg protein/ml) was diluted to 6 ml with buffer and divided equally into two cuvettes. DOC (240 PM) was added to the experimental cuvette, and the difference spectrum, Curve A (solid line), was recorded. Sodium malate (5 mM) was then added to the contents of both cuvettes, and after exhaustion of oxygen by the mitochondria in both cuvettes, Curve B (dashed line) was recorded. A second aliquot of mitochondria was diluted and treated in a similar manner except for the addition of succinate as substrate. The spectral change caused by the presence of DOC with succinate treated anerobic mitochondria is shown by Curve C (dotted line).
added to both cuvettes to initiate respiration, The appearance of an absorption band at 442 rnp indicated that oxygen had been exhausted first in the experimental cuvette containing DOC, followed by the subsequent disappearance of the absorption band of cytochrome oxidase at 442 rnp, showing that oxygen had been exhausted via the respiratory chain in the mitochondrial suspension present in the reference cuvette. The difference spectrum (curve B) was then recorded, showing directly the presence of the DOCinduced spectral trough in the anerobic sample. Trace C was bbt’ained in a similar manner, but succinate rather than malate was used as substrate. Clearly the absorption
decreases due to the presence of DOC remained with oxygen absent from the sample, although there were some displacements in the location of the minima. The difference in location of the absorption bands resulting from the presence of DOC in the aerobic or anerobic sample are similar to those described previously (35, 39) for substrate interaction of drugs with oxidized and reduced cytochrome P-450 of liver microsomes. When a similar series of experiments was carried out, using sodium dithionite as reductant, the spectral change associated with DOC interaction was obliterated, suggesting that chemical reduction with sodium dithionite, in contrast to enzymic reduction via the
SPECTRA
OF ADRENAL
cytochrome P-450 respiratory chain, markedly modified DOC interaction with cytochrome P-450. Spectral properties of the cytochrome P-450. CO complex. Cytochrome P-450 was first recognized by the large absorption band appearing at 450 mp after gassing a sample of the reduced pigment with carbon monoxide. Although cytochrome P-450 has been described as a component of microsomes from a number of different organs as well as a component of adrenal cortex mit’ochondria (cf. Fig. 2 and Refs. 2, 18, 19, 30), there have been no reports on associated spectral changes in the visible part of the spectrum. A series of spectral studies was therefore carried out with adrenal cortex mitochondria to establish t#he nature of t,he CO complex of cytochrome P-450 in this part of the spectrum. Fig. 6 (curve R) demonstrates that CO gassing of a sample of adrenal cortex mitochondria pretreated with succinate, cyanide, and DOC, followed by malate oxidation to establish anerobiosis, results in a displacement of the absorbance difference (cf. curve A) but no obvious format’ion of any new absorption bands. It is only when the spectral difference between the CO gassed anerobic sample and the malatetreated, non-CO gassed anerobic sample is examined t,hat the absorption properties of t,he CO complex of cyt,ochrome P-450 are detected. As shown in Fig. 6, curve C, rather broad absorption maxima at about 570 and 545 mp are observed. These spect)ral studies also revealed the presence of a weak absorption band with a maximum at about 647 rnp which disappears when the sample becomes anerobic during malate oxidation. This spectral change is associated with the appearance of t,he 555 m/l absorption maximum of reduced cytochrome P-450. Since cytochrome P-450 can be reduced eneymically either in the presence or absence of hydroxylatable substrate (DOC) and by addition of tricarboxylic arid cycle substrates such as succinate, malate, isocitrate, etc., spectral studies were carried out to determine whether there was any variation in the pattern of the spectral properties of the CO complex of reduced cyt,ochrome P-450. As shown in Fig. 7, the magnitude of the absorption bands with maxima at about 575
CORTEX
\ -006
743
MITOCHONURIA
/
i
I
B Malate (onoerobvz) + CO Minus Oxldlzed C Molate (anaerobic)+ CO Minus Malate kmaeroblc)
I”“i”“l”“I”l’
500
550 Wavelenqth
600
650
cm+)
FIG. 6. The effect of CO on the visible absorption of reduced cytochrome P-450. A 0.5.ml aliquot of adrenal cortex mitorhondria (44 mg/ml) R~S diluted to 6 ml wit,h isotonic bufier. Potassium cyanide (1 mM), sodium sllcciuate (5 mn~), and I>OC (0.24 mM) were added to the mixtrue, and the sample was divided eqllally ink) two cllvettes. After est,ablishment of a baseline of eqllal light absorption, sodium malate (5 mv) was added to one cuvette (experimental) aud the sample was permitted to ntilize the oxygen dissolved in the react.ion medium. The differenre spectrrrm of reduced cgtochrome P-450 (CIIIVC A) was then recorded. The contents of the anaerobic sample were theu gassed for 30 seconds with CO and the spectral changes recorded (Curve B). An aliqllot of sodirlm malate (5 mM) was t)heu added to the contents of the reference cllvette, alld, after attaining anaerobiosis, the difference spectrlml (Cltrve C) of the rcdrlced cytochrome P-45O’CO complex minrls reduced cytochrome P-450 was recorded.
and 545 rnp varied, depending on the conditions employed. Examination of t,he spectral region from 400 to 500 rnp revealed no discernible difference in the spectral properties of the cytochrome P-45O.CO complex. It appears that the presence of DOC favors the spectral changes associated with the formation of cytochrome P-450 ‘CO complex at 575 rnp, and the absence of hydroxylatable substrate favors the formation of a compound with a spectral maximum at about 545 rnp. These differences may be correlated with the possible change in the strength of ligand binding occurring under t.he various conditions examined. Correlation to the spect:ral properties of cytochrome P-450 of liver microsomes. Previous reports (18, 42) have demonstrat’ed that
744
CAMMER AND ESTABROOK
A Swcwwie(Anambic~+ CO MINIS SuccinotdAnaerobic) E Succ~nots(Amembic)+DOC+CO Mmur S~~~c~nols(Ar.wcbiil+DOC C Succimfe+KCN+Moi~klA~robicl+CO Minus Succirmie+KCN+M~late(Anoerobicl I 490
I I I I r I, 520 550 560 Wovelength
I, 610
I I ,I 640
FIG. 7. The spectral properties of the reduced cytochrome P-45O.CO complex obtained with varying substrates in the presence and absence of DOC. Experiments were carried out as described in Fig. 6. For Curve A, the diluted adrenal cortex mitochondria in the absence of DOC and cyanide were keated with sodium sucoinate (5 mM) and the anerobic sample in the reference cuvette was gassed for 30 seconds with CO. The difference spectrum presented in Curve B was obtained with diluted adrenal cortex mitochondria, to which DOC (0.24 mM) and sodium succinate (5 mM) were added prior to gassing with CO. The difference spectrum of reduced cytochrome P-45O.CO complex observed in the presence of potassium cyanide (1 mM), sodium succinate (5 mM), and sodium malate (5 mM) in the absence of DOC is shown in Curve C.
sodium dithionite reduces a pigment of liver microsomes not reduced during the aerobic steady state by DPNH. Mason et al. (20) have also demonstrated the enzymic reduction of a similar pigment by TPNH under anerobic conditions. To determine whether these spectral changes may represent the spectral contribution of reduced cytochrome P-450, experiments of the type presented in Fig. 8 were carried out. For these experiments microsomes were obtained from livers of rabbits treated with phenobarbital (43) to cause maximal spectral change on reduction of cytochrome P-450. Curve A of Fig. 8 illustrates the spectral contribution of cytochrome bgof liver microsomes reduced during the aerobic steady state by TPNH. No significant difference was observed whether TPNH or DPNH was used as reductant. The subsequent addition of N&z04 to the sample (curve B) caused a pronounced increase in absorbance at about 556 rnp, to-
gether with the appearance of a trough at about 576 mp. When the difference spectrum is measured between a sample treated with Na&04 to reduce both cytochrome bs and cytochrome P-450 and a sample treated aerobically with TPNH or DPNH, the difference spectrum of reduced minus oxidized cytochrome P-450 (curve C) is obtained. A similar type of spectral change can also be obtained when TPNH rather than NazSz04 is added to an anerobic sample, and confirms the earlier observation of Mason et al. (20) and the recent report by Nishibayashi and Sato (44). The shape of the spectral change associated with reduction of cytochrome P-450 of rabbit liver microsomes is remarkably similar to that observed upon reduction of cytochrome P-450 of adrenal cortex mitochondria (cf. Figs. 1, 3, 4, and 6). The subsequent addition of CO to the anerobic sample of rabbit liver microsomes results in the formation of two rather weak absorption bands with maxima at about 545 and 575 rnp comparable with that described in Fig. 6 for the spectral contribution of cytochrome P-450. CO complex of adrenal cortex mitochondria. Extinction coeJ.kients of cytochrome P-&O. The experiments described above now permit the identification of spectral changes, as observed by difference spectrophotometry, associated with cytochrome P-450. The millimolar extinction coefficient of the reduced cytochome P-450. CO complex with a maximum at 450 rnp has been determined by Omura and Sato (19) to be 91 rnr’ cm-‘. This extinction coefficient was used as the basis for determining the content of cytochrome P-450 in a sample, and the change in extinction coefficients occurring upon reduction of cytochrome P-450 and the complexing of reduced cytochrome P-450 with CO was determined (Table 11). Of interest is the relatively low extinction change occurring at 432 rnp on reduction of cytochrome P-450. The very low extinction change observed in the visible portion of the spectrum upon formation of the CO complex of reduced cytochrome P-450 explains the failure to detect these absorption bands in previous experiments. A comparison~_of the _ changes in extinction observed with adrenal cortex
SPECTRA TABLE
OF AURENAL
II
EXTINCTION CH.LNGES OCCURRINO DURING CYTOCHROME P-450 REDUCTIONOR~NTER.4CTION WITH SURSTRaTE" Condition
\Vavt>h
A(m1.0 cm-l)
432-490 555-576 420-490 573-600 450-490 570-59
24 10 30 4 91 2.5
Anerobic minns aerobic Aerobic + DOC mintis aerobic Anerobic + CO minus anerobic
a Experiments were carried out as described in Figs. 3-6. In each experiment the content of cytochrome P-450 was determined from the absorbance change at 450 rnr relative to 490 rnp observed after rediiction with Na&04 and gassing with CO, employing the extinction coefficient recommended by Omura and Sato (19). Comparable results have beeu obt)ained with microsomes from rabbit liver.
CORTEX
715
MITOCHONDRIA
certain whether cytochrome P-450 may exist in two forms in adrenal cortex mitochondria or liver microsomes (38). One form predominates in the presence of a hydroxylatable substrate, such as DOC, while the other occurs in the absence of substrate. The difference between the two forms may reflect an alteration in Iigand binding to the heme of cytochrome P-450 as described previously (38, 39, 41), or it may result from monomer to dimer transitions of the hemoprotein. These two forms are observed by the spectral changes occurring in the presence and absence of DOC during the experiments with adrenal mitochondria. In many instances these differences are smal1 and difficult to detect, but in other instances, such as the 555
I
/
1
;: ,!
mitochondria and those observed with microsomes from rabbit liver indicates a very close correlation for the two types of systems. DISCUSSION
The present studies have attempted to define the spectral changes occurring upon reduction of pigments associated with substrate oxidation by beef adrenal cortex mitochondria. These mitochondria are of interest be&use they contain not only the “normal” respiratory chain of mitochondria functional in the synthesis of ATP but also a second type of respiratory chain containing cytochrome P-450 functional in the hydroxylation of DOC and cholesterol. Quantitatively, adrenal cortex mitochondria contain a complement of cytochromes a + u3, b, and c + cl very similar to that reported for liver mitochondria. As reported by Harding and Nelson (2), the preponderant pigment of these mitochondria is cytochrome P-450. Utilizing inhibitors of the normal respiratory chain, it was possible to assess the spectral changes occurring during reduction of cytochrome P-450 and its interaction with DOC. From these studies the tentative scheme presented in Fig. 9 was constructed. The wavelengths designated indicate the absorption maxima or minima observed upon transition from one state to another. It is un-
I
I
490
I
I
I
520
r
8,
/
550
I,
II,
580
I
610
I
I
640
I
/
,
660
Wavelength(mp1
FIG. 8. The difference spectrum of reduced cytochrome P-450 of rabbit liver micrdsomes. A 0.6.ml aliquot (30 mg protein/ml) of mi&osomes prepared from livers of rabbits pretreated for 5 days by daily injections @Omg/kg) of phenobarbital was diluted to 6 ml with 50 mM Tris buffer, pH 7.4. The contents of the diluted sample were divided equally into two cuvettes and a baseline of equal light absorption was recorded. A 0.2.ml aliquot of a TPNII solution (20 mM) was added to the contents of the experimental cuvette, and the difference spectrum of reduced cytochrome bs (Curve A) obtained during the aerobic steady state was recorded. A few crystals of solid sodium dithionite were then added to the contents of the same cuvette, and the difference spectrum (Ciirve B) was recorded. After addition of a 0.02-11~1aliquot of a TPNH solution (20 mM) to the contents of the reference cuvette to reduce cytochrome b,, the difference spectrum (Curve C) of redrlced minus oxidized cytochrome P-450 was recorded.
746
CAMMER
AND
ESTABROOK
P-450 Fe”,DOC Cb
Co$os$rone
2 FIG. 9. Schematic representation of possible oxidation and cytochrome P-450 in the presence or absence of deoxycorticosterone
visible spectral changes occurring upon formation of the CO complex of reduced cytochrome P-450, the spectral properties of the pigment can be markedly modified. The ability to now measure the spectral changes occurring upon reduction of cytochrome P-450 permits experiments to evaluate directly the interaction of oxygen with this pigment during hydroxylation reactions. It is apparent from the studies presented here that cytochrome P-450 is largely in the oxidized form during the aerobic steady state of hydroxylation when adrenal cortex mitochondria or liver microsomes are employed. The kinetic transitions occurring during the oxidation and reduction of cytochrome P-450 will be described in a subsequent paper. In the present study, no evidence was found which could argue in favor of or against an oxygenated form of ferrous cytochrome P450. Also, since the present studies employed turbid suspensions of mitochondria or microsomes and therefore have been restricted to spectral changes assessed only by difference spectrophotometry, it has not been possible
reduction changes of (DOG), CO, and OZ.
to assign absorption maxima to the ferric form of the hemoprotein. However, it has been possible to determine quantitatively the cytochrome content of adrenal cortex mitochondria and to assign changes in extinction occurring during the oxidation and reduction of these pigments. REFERENCES 1. CAMMER, W., AND ESTABROOK, R. W., Arch. Biochem. Biophys. 122, 721-734 (1967). 2. HARDING, B. W., AND NELSON, D. H., J. Biol. Chem. 241, 2212 (1966). 3. PURVIS, J., BATTU, R., AND PERON, F. G., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 4. KORITZ, S. B., Biochem. Biophys. Res. Commun. 23, 485 (1966). 5. MCCARTHY, J. L., AND PERON, F. G., Biochemistry 6, 25 (1967). 6. NAKAMURA, Y., OTSUKA, H., AND TAMAOKI, B. I., Biochim. Biophys. Ada 122, 34 (1966). 7. SWEAT, M. L., J. Am. Chem. Sot. 73, 4056 (1951). 8. HAYANO, M., AND DORFMAN, R. I., J. Biol. Chem. 211, 227 (1954).
SPECTRB
OF ADRENAL
9. Ha~axo,hl., LINUBERG, &I. C., DORFMSN, R. I., HANCOCK, J. E. H., .\XD DOEKING, W. VON E., .4rch,. Biochem. Riophys. 59, 529 (1955). 10. ESTABROOK, R. W., COOPER, 11. Y., AND ROSENTHAL, O., Hiochem. Z. 383,741 (1963). 11. COOPER, 1). Y., LEVIN, S., NARASIMHULU, S., ROSENTHAL, O., AND EST.~BROOK, R. W., Science 147, 400 (1965). 12. H.\RDING, B. W., WONG, S. H., AND NELSON, D. II., B&him. Biophys. Beta 92, 415 (1964). 13. WILS~X,L. D.,NELsoN,D. H., AND HARDING, B. W., Biochim. Biophys. ;lcta 99,391 (1965). 14. COOPER, I). Y., NAR.~SIMHULU, S., HL.~DE, A., XIICH, W., FOHOFF,~., ANT) ROSENTH~L,~., Life 67-i. 4, 2109 (1965j. 15. CAMMER, W., COOPER, D.Y., AND EST.~BROOK, 1~. W., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (19G7). 16. HARDING, B. W., OLUHAM,S. B., ANU WILSON, L. I>., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 17. SIMPSON, E. It., .\ND BOYD, G. S., Biochem. Biophys. Res. Commun. 24,lO (1966). 18. COOPER, D. Y., NIRASIMHULU, S., ROSENTHAL, O., AND EST;ZBROOK, R. W., in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 841, Wiley, New York (1965). T., .~PI‘DSATO, R., J. Biol. Chem. 233, 19. OMURA, 2270 (1964).20. MASON, H. S., NORTH, J. C., AND VANNESTE, M., Federation Proc. 24,1172 (1965). 21. SPIRO, M. J., .~ND BALL, E. G., J. Biol. Chem. 236, 231 (1961). 22. RINOSHITB, T., HORIE, S., SHIMAZONO, N., .~ND YOHRO, T., J. Biochem. (Tokyo) 60, 39 (1966). 23. HoRIE,S.,KINOSHITA,T.,.~NDSHIMAZONO,N., .I. Biochem. (Tokyo) 60,660 (1966). 24. KI~~uR.?, T., in “Biological and Chemical Aspects of Oxygenases” (K. Bloch and 0. Hayaishi, eds.), p. 179. Maruzen Co. Ltd., Tokyo (1966). 25. CHANCE, B., Methods Enzymol. 4,273 (1957). 2fj. CORNALL, A. G., BARDAWILL, C. J., AND DAVID, h1. M., J. Biol. Chem. 177,751 (1949). 27. CAMMER, W., AND ESTABREOK, It. W., Federation Proc. 25, 281 (1966). 28. GRANT, J. K., .\ND BROWNIE, A. C., B&him. Biophys. Acta 18, 433 (1955). 29. S~~EAT, 1LI. L., ANI) LIPSCOMB, M. I~., J. Am. Chem. Sot. 77, 5185 (1955). 30. OMURA,T., s.4~0, R., COOPER,D.Y.,ROSENTHBL, O., AND ESTABROOK, R. W., Federation Proc. 24, 1181 (1965).
CORTEX
MITOCHONl)l~IA
747
31. EST.~IIROOK, R. W., SCHENKMIN, J. B., CAMMER, W., REMMER, H., COOPER, 1). Y., N.\R,~SIMHULU, S., .\ND ROSENTH.~L, O., in “Biological and Chemical Aspects of Oxygenases” (K. Bloch and 0. Hayaishi, eds.), p. 153. Maruzen Co. Ltd., Tokyo (1966). 32. ESTABRO~K, 1~. W., .IND HOLO~INSKY, A., J. Biophys. Biochem. Cytol. 9, 19 (1961). 33. ESTBBROOK, R. W., AND M.~ITR.~, P. K., dnd. Biochem. 3, 369 (1962). T., SANDERS, E., COOPER, I). Y., 34. OMURA, ROSENTHAL, O., ABD Es~an~ooK, 1~. W., in “Non-Heme Iron Proteins: Role in Energy Conservation” (A. San Pietro, ed.), p. 401; Kettering Symposilun. Antioch Press, Yellow Springs, Ohio (1905). 35. CMURA, T., CAMMER, w., COOPER, D. Y., ROSENTHAL, O., AND ESTABROOK, It. W., in “Elektrochemische Methoden und Prinzipien in der Molkealar-Biologie” (H. Berg, ed.), p. 25; Third Jaener Symposium. Akademie-Verlag, Berlin (1966). 36. KLINGENBERG, M., in “Fnnktionelle und Morphologische Organisat,ion der Zelle” (P, Karlson, ed.), p. 69. Springer-Verlag, Berlin (1963). 37. NARASIMHULU,S.,COOPER, D.Y., ANDROSENTHAL, O., Life Sci. 4, 2101 (1965). 38. COOPER, D. Y., NIRASIMHULU, S., ROSENTHAL,~., AND EsT.~BROOK, R. W.,in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 39. REMMER, H., &HENKMSN, J. B., ESTABROOK, R. W., SASAME, H., GILLETTE, J., NARASIMHULU,S.,COOPEK, D.Y., AND ROSENTHAL, O., Mol. Pharmacol. 2, 187 (1966). 40. IMAI, Y., AND SATO, li., Hiochem. Biophys. Res. Commun. 23, 5 (1966). 41. ESTABROOK, 13. W., PCHENKMAN, J. B., CurMER, W., COOPER, D. Y.,NARASIMHULU, S., S., .\NI) ROSENTH.\L, O., in “Hemes and Hemoproteins,” (B. Chance, R. W. Estabrook, and T. Yonetani, eds.), p. 511. Academic Press, New York (1967). 42. S.4~0, R., OMURA, T., .\ND NISHIBAYASHI, H., in “Oxidases and Related Redox Systems” (T. E. King, 1-I. Mason, and M. Morrison, eds.), p. 861. Wiley, New York (19G5). 43. REMMER, H., GREIM, II., SCHENKMAK, J. B., AND ESTABROOK, R. W., in “Methods in Enzymology,” Vol. 10 (“Oxidation and Phosphorylat,ion”; R. W. Estabrook and M. PulIman, eds.), p. 703. Academic Press, New York (1967). 44. NISHIBAYASHI, H., AND 89~o,R., J. Biochem. 61, 491 (1967).
OF
BIOCHEMISTRY
AND
Spectrophotometric
BIOPHYSICS
122, 735-747 (1967)
Studies Cortex
WENDY Departtrlent
CAMMER2
of the
Pigments
of Adrenal
Mitochondria’ RONALD
AND
of Biophysics rind Physical Cnioer,sity of Pennsylvania, lteceived
Biochemistry, Philadelphia, August
W. ESTABROOKS Johnson Research Foundation, Pennsylvania 19104
8, 1967
St,rldies have been carried out to determine the spectral changes occurring during srlccinate and malate oxidation by mitochondria from beef adrenal cortex. The conThe spectral tent of cytochromes a + a3 , b, c + cl , and P-450 have been determined. propert.ies of redllced cgtochrome P-450, its complex with CO, and the interaction of oxidized cytochrome P-450 with deoxycorticosterorle have been determined. as well as the location of absorption The changes in extinction coefficients, maxima and minima occrlrring dluing cptochronle P-450’s oxidation arid redrlctioll associated with hydroxylatiorr of LNC, are described. The results obtained are conpared with those for cytochrome P-450 of microsomes prepared from rabbit liver.
adrenal cortex mitochondria (2, 12) and has been implicated in the lip-hydroxylase rea&on (13-16) as well as in t’he conversion of cholesterol to pregnenolone (4, 17). Spectral studies of reduced and oxidized cytochrome P-450 in microsomes are complicated by interference from cytochrome bg (18), and attempts to isolate cytochrome I’-450-containing fractions from mi~rosomal membranes have led to obvious alterations in its spectral propertries (19-21). Recently a cytochrome I’-450-enriched fraction has been obtained (14) from adrenal cortex mitochondria, and permitt’ed the measurement (14, 22, 23) of some of the spectral properties of cytochrome P-450. To assess the possible alterations occurring to cytochrome l’-450 during its isolation, the spect)ral studies of adrenal cortex mitochondria reported here were undertaken. In addition t’he present study examines the changes of cytochrome l’-430 occurring during steroid hydroxylation, and confirms (2) the presence of a normal mitochondrial respiratory chain in adrenal cort,ex mitochondria. These studies also support the increasing body of evidence for an interact’ion in adrenal cortex mitochondria between a normal respiratory chain
Nit,ochondria isolated from the cortex of the beef adrenal gland carry out the reactions of oxidative phosphorylation characteristic of mitochondria isolated from heart,, liver, kidney, etc. (1). In addition these mtochondria also carry out certain steroid hydroxylation reactions (2-6), most not’ably the 1I@-hydroxylation of deoxycorticosterone (7, 8). The lip-hydroxylase reaction of adrenal cortex mitochondria has been shown to resemble ot,her mixed function oxidations in incorporatjing 180 from molecular oxygen (9) into the subst’rate molecule (deoxycorticosterone, DOC) and in t,he stoichiometry for oxygen and DOG (1). Furthermore, cyt’ochrome P-450, which is functional in many microsomal mixed function oxidations (10, II), has been shown to be present, in 1 These stIltlies were supported in part by Granls from the U.S. Public Health Service ((+M 12202) and the National Science Folmdatioll (GE 2451) . 2 Predoctoral Fellow of t,he GradLlate Group on Molewdar Biology; slIpported by U.S. Public IIealth Service grant 5 TO1 GM-00694.07. 3 These stlldies were carried ollt, drlrilrg the tenrlre of a U.S. Prlblic Health Service Research Career Development Award (GM-K3-4111). 735
736
CAMMER
AND
containing cytochromes a, u3, b, and c + cl and a second type of respiratory chain leading to a steroid hydroxylation and containing cytochrome P-450 (2, 3, 15, 16, 24). METHODS The preparation of beef adrenal cortex mitochondria has been described previously (1). For the experiments reported here, the isotonic bluffer employed contained 0.25 M sucrose, 20 mM KCl, 10 mM potassium phosphate buffer, pH 7.4, 15 mM triethanolamine chloride buffer, pH 7.4, and 5 rnM MgC12. Sodium succinate and sodium malate were prepared as 1 M solutions which had been neutralized to pH 7. Deoxycorticosterone (DOC) was obtained from the Sigma Chemical Company, St,. Louis, Missouri, and was prepared as a 48 mM solution in ethanol. The wavelength scanning recording spectrophotometer has been described by Chance et al. (25). Protein determinations were carried out by the method of Gornall et al. (26). RESULTS
Cytochromes of adrenal cortex mitochondria. It has been established that Krebs’ tricarboxylic acid cycle substrates are capable of supporting the lip-hydroxylation of DOC by reducing the endogenous mitochondrial pyridine nucleotides (2,3,27-29) and thence cytochrome P-450, via a flavoprotein and a nonheme iron protein (30, 31). When succinate is used as substrate, reduction of these electron carriers, including cytochrome P-450, is prevented by inhibitors of the normal mitochondrial respiratory chain. Accordingly it was considered possible to examine spectrophotometrically the pigments reduced when succinate was used as substrate in order to reduce cytochrome P-450 as well as the normal respiratory chain cytochromes of adrenal cortex mitochondria, and to compare this result with spectra obtained when succinate was added in the presence of cyanide, where only the cytochromes of the normal respiratory chain and not cytochrome P-450 were reduced, i.e., inhibition of the normal respiratory chain prevents the formation of malate from succinate or the generation of energy required for the succinate-linked reduction of pyridine nucleotides, and thereby prevents reduction of cytochrome P-450. In order to obtain the difference spectra
ESTABROOK
shown in Fig. 1, a suspension of adrenal cortex mitochondria was diluted in isotonic buffer and divided equally between two cuvettes, and the spectra were recorded with a wavelength scanning recording spectrophdtometer. Trace A was obtained after the addition of cyanide and succinate to the experimental cuvette. The absorption bands with maxima at 605 and 445 rnp, 561 and 428 rnp, and 552 and 419 rnp are characteristic of the normal respiratory chain cytochromes (a + u3), b, and (c + cJ, respectively. From spectral studies of this type the concentrations of normal respiratory chain cytochromes in adrenal cortex mitochondria were determined, and the results are summarized in Table I. The values obtained with adrenal cortex mitochondria agree quite well with the cytochrome content of liver mitochondria (30). In contrast, the cytochrome content of beef adrenal cortex mitochondria reported by Harding and Nelson (2) are considerably higher than those found here. In the experiments of Harding and Nelson, this discrephncy can be attributed to interference from the spectral contributions of cytochrome P-450 (see below). The contribution of cytochrome P-450 leads to deceptively high values for cytochromes b and c, as illustrated by the difference spectrum (curve B) presented in Fig. 1. In this experiment an aliquot of beef adrenal cortex mitochondria was diluted as described above, and succinate was added as substrate in the absence of cyanide. After a brief period of incubation, an increase in absorbancy at 445 rnp indicated reduction of cytochrome a3 associated with exhaustion of oxygen in the reaction cuvette. The spectrum obtained under these conditions is similar to that reported by Harding and Nelson (2). Spectrum of cytochrome P-450. In order to determine the contribution of those pigments reduced by succinate in the absence of cyanide relative to those pigments reduced by succinate in the presence of cyanide, the difference spectrum presented in Curve C of Fig. 1 was recorded. This spectrum represents the difference spectrum of reduced cytochrome P-450 minus oxidized cytochrome P-450. A broad a-band with a
WECTRA
OF ADRENAL
CORTEX
MITOCI-IONDRIA
428 /
28 ,445
Succinote+ KCN Minus Oxidized B --- Succinate(onoerobic1 Minus Oxidized C..... Succinote(onoerobic) Minus Succinote+ KCN
A-
I 1 Wovelength
I
I
I
, I 550
I
I,,
I 600
I
I
I
, I 650
FIG. 1. Spectral changes of adrenal cortex mitochondria occurring during cytochrome reduction with succinate as substrate. Adrenal cortex mitochondria were diluted to 6 ml with bluffer mixture and divided equally into two cuvettes. Succinate (5 mM) and potassium cyanide (1 rnw) were added to one cuvette, and the difference spectrum shown in Curve A (solid line) was recorded. An equivalent dilution of mitochondria was divided equally in two cuvettes, and succinate (5 mM) was added in the absence of cyanide. After utilization of the oxygen dissolved in the reaction medium had occurred, Curve B (dashed line) was recorded. Succinate (5 mM) and potassillm cyanide (1 mM) were then added to the other cuvette (referenre cuvette), and the difference spectrum shown in Curve C (dotted line) was recorded. The visible spectra were obtained at a protein concentration of 3.7 mg/ml, while the Soret spectra were recorded with a protein concentration of 1.7 mg/ml.
maximum at about 555 mp and a Soret band with a maximum at 432 rnp are observed. In beef adrenal cortex mitochondria the spectral contributions of cytochrome P-450 are significantly greater than those of cytochrome b and c + cl of the normal respiratory chain. The positions of the maxima for the spectral contribution of reduced minus oxidized
cytochrome
P-450 agree well
with those found by Cooper et al. (18) using adrenal cortex microsomes. While recording these spectra, it was noted that there frequently was a significant decrease in absorbance not attributable to a change in the oxidation reduction of a cytochrome pigment. This is illustrated by the negative displacement from the baseline in
Fig. 1, curves B and C. This decrease in absorbance could be caused either by a swelling of mitochondria in the presence of substrate or to the spectral changes associated with reduction of the flavoprotein and the nonheme iron protein functional in the electron transport system from TPNH to cytochrome P-450. The large decrease in absorbance at about 460 m,u, as well as the relatively high content of nonheme iron protein as determined by electron paramagnetic spectroscopy (Table I), suggest that the wavelength independent loss of absorbance in the visible region of the spectrum is possibly due to reduction of flavoprotein and nonheme iron protein. Previous studies (1, 12, 28) have shown
738
CAMMER
AND
ESTABROOK
TABLE CONTENT
OF
RESPIRaTORY
ClRRIERS
I
OF
ADRENAL
CORTEX
MITOCHONDRL~” Content
Pigment
Cytochrome a + a3 b c -t Cl P-450 Nonheme iron TPN + TPNH DPN + DPNH
Method
Spectrophotometric
Spectrophotometric wavelengths 6OFG-625mp 562-575 mp 551-540 mu 45C490 rnfi EHR spectrometry Enzymic assay Enzymic assay
(mab : cm-l)
16 20 19 91
Ad&al mitochondria
0.23 0.17 0.29 1.5 2.6 2.8 6.3
(mjmoles/mg
protein)
Adrenal mitochondria
Liver mitochondria
0.756 0.32’ 0.67’ 1.35
0.22( O.lW O.Xlc
2.gd 5.5d
a Respiratory chain cytochromes were determined from difference spectra of the reduced minus oxidized pigments using the extinction coefficients indicated (32). Cytochrome P-450 was determined from the magnitude of the absorption band of the reduced cytochrome P-450 carbon monoxide complex using the extinction coefficient determined by Omura and Sato (19). Pyridine nucleotides were determined by enzymic assay (33), and non-heme iron was determined by comparing the magnitude of the g = 1.94 electron paramagnetic resonance signal in dithionite treated mitochondria with the signal obtained from a known amount of non-heme iron protein purified from adrenal cortex mitochondria (34, 35). * Data from Harding and Nelson (2). c Data from Estabrook and Holowinsky (32). d Data from Klingenberg (36).
that malate, as well as succinate, is a suitable substrate for supporting the maximal rate of DOC hydroxylation by adrenal cortex mitochondria. Hydroxylation occurring in the presesence of malate is relatively insensitive to cyanide when compared wit’h succinate-supported hydroxylation. When spectra were recorded with malate as substrate, t,he difference spectra presented in Fig. 2 were obtained. In the aerobic steady state (curve A) a part’ial reduction of cytochromes was observed. The reduction of flavoprotein and nonheme iron protein is ascertained by the pronounced decrease in absorbance with a minimum at about 460 rnp. After a few minutes an increase in absorbance occurred at 445 rnp, indicating reduction of cytochrome a3 and attainment of anerobiosis. The difference spectrum of malate-treated mitochondria in the anerobic state minus mitochondria in the oxidized st,ate, where malate had not been added, is shown by curve B of Fig. 2. This spectrum is similar to that shown in Fig. 1, curve B, where succinate was used as substrate. It is apparent that malate as well as succinate
are capable of reducing not only the normal respiratory chain cytochromes but also cytochrome P-450. This conclusion is supported by the spectrum presented in curve C of Fig. 2, where the malate-treated mitochondria in the anerobic state were gassed briefly with carbon monoxide. An intense absorption at about 449 rnp (note difference in scale) characteristic of the reduced cytochrome P-450 carbon monoxide complex was observed. In agreement with earlier studies of Harding and Nelson (a), t’he content of cytochrome P-450 (cf. Table I) is sufficiently high to obscure the absorbancy contributions of the other cytochromes present in adrenal cortex mitochondria. The presence of CO interaction with cytochrome oxidase is indicated, however, by the small decrease in absorbancy at 604 rnp concomitant wit,h the appearance of a shoulder at about 595 rnp. Note also the increase in absorbance of the broad complex absorption band at about 552 rnp. DOC-Induced spectral changes. Further studies were carried out attempting to establish the changes in the oxidation state of
SPECTRA A,B
OF ADRENAL
CORTEX
739
MITOCHONDRIA
C
0.16+0.2
0.08--0.1
(oeroblc)-
B. Molote (onaerobic)-Oxidized
-0.24---0.3
1
350
Oxidized
Minus Oxidized
, I
I,,
I
400
I
I
I,
I
I
I
I,
450
I
500 Wavelength
-0.06
I
I
I
,
550
I
I
I
I,
I
I
I
600
(mp)
FIG. 2. Spectral changes of adrenal cortex mitochondria associated with malate oxidation. Bs in Fig. 1, an aliquot of adrenal cortex mitochondria was diluted to 6 ml (4.5 mg protein/ml) and divided equally int,o two cuvettes. Sodium malate (5 mM) was added to one cnvette, and the difference spectrum of pigment changes during the aerobic steady state was recorded, Cllrve A (dotted line). After lttilization of oxygen in the reaction medium, the difference spectrum, Curve B (solid line), was recorded. Carbon monoxide was bubbled for about 1 minute into the contents of t,he cuvette and the difference spectrum, and Curve C (dashed line) was recorded. Note: The spect,ral changes in the region 38s490mr for Curve C were recorded with a less sensitive absorbance scale.
cytochrome P-450 without interference from absorption bands of t’he normal respiratory chain cbytochromes. In order to obtain t,he spectra presented in Fig. 3, a baseline of equal light absorbance was recorded using a sample of diluted mitochondria where the respiratory chain cytochromes were previously reduced by the addition of cyanide and succinate. Addition of the ll&hydroxylase substrate deoxycorticosterone (DOC) to the experimental cuvette resulted in decreases in absorbancy (curve A, Fig. 3) at 570 and 535 rnp, accompanying the spectral change at’ 420 rnp (14) characterist,ic of substrate interact,ion wit,h microsomal cytochrome P-4*50of these mixed funct’ion oxidase
systems reacting with steroids (37, 38) or drugs (39-41). Further addition of malate to the experimental cuvette (curve B) altered this difference spectrum only slightly, but when the llfl-hydroxylase reaction had exhausted the oxygen (1) in the experiment,al cuvett’e (curve C), one observes, as in Figs. 1 and 2, the appearance in the difference spectra of absorption bands at 553 and 433 rnp which correspond to t’he difference spectrum of the reduced form of cytochrome P-450 (cf. also Fig. 1, curve C). Of interest is the observation that the absorption band with a maximum at 432 rnp is of low absorbance relative to the baseline, which suggests the persistence anerobically of the sb-
740
CAMMER
AND
ESTABROOK
386
.06
-.06-.08
-1
575 A- - DOC minus Oxidized B ..‘... DOC Plus Malate Steady State Minus Oxidized DOC Plus Malate Anaerobic CMinus Oxidized D-- DOC Plus Malate + Na2S204 I, I I I I, I I I I
500
550
,
1 I
I
600
Wavelength (mpL)
FIG. 3. The effect of deoxycorticosterone (DOC) on pigment reduction with malate as substrate. Adrenal cortex mitochondria were diluted in buffer to 6 ml; sodium succinate (5 IIIM) and potassium cyanide (1 mu) were then added, and the suspension was divided equally into two cuvettes. The mitochondrial protein concentrations were 4.5 mg/ml for the spectra recorded from 480 to 630 rnp and 2.0 mg/ml for spectra recorded from 370 to 486 rnp. The difference spectrum shown by Curve A (dashed line) was obtained after adding DOC (240~~) to the contents of one cuvette. Sodium malate was then added, and pigments reduced aerobically during malate oxidation were recorded (Curve B, dotted line). After utilization of oxygen in the reaction cuvette, Curve C (solid line) was recorded. The subsequent addition of sodium dithionite to the anerobic sample results in a further reduction of the pigments as shown in Curve D.
sorbancy change associated with DOC addition (see below). The subsequent addition of sodium dithionite (curve D) results in further increase in absorbance at 432 mp. This further change is attributable to the loss of the DOC-induced spectral contribution with a minimum at 420 mp. To determine the contribution of DOC to the absorbance change, a similar series of experiments (Fig. 4) was carried out on a diluted mitochondrial suspension which had been treated with cyanide and succinate in order to reduce the normal respiratory chain cytochromes as described above. In agree-
ment with the results presented in Fig. 3, curve A, the addition of DOC to the experimental cuvette (trace A) showed decreases in absorbance at 570 and 421 rnp. The subsequent addition of DOC to the reference cuvette restored the baseline of equal light absorbance. The further addition of malate to the experimental cuvette (trace B) resulted in only a partial reduction of cytochrome P-450, as evidenced by the small absorption bands at 555 and 422 rnp. On exhaustion of the oxygen present in solution in the experimental cuvette (trace C), one again sees the appearance of absorption
SPECTRA
OF ADRENAL
CORTEX
MITOCHONDRIA
741
KCN Plus Succ Both A-DOC Mmus Oxldlred B.,,.... DOC Plus Malate Steady State Minus DOC CDOC Plus Malate Anaerobic
Wavelength
(mpL)
FIG. 4. The spectral changes of cytochrome P-459 during malate oxidation in the presence of DOC. Adrenal cortex mitochondria were diluted in buffer to 6 ml; sodium succinate (5 mM) and potassium cyanide (1 mM) were added, and the sample was divided equally into two cuvettes. The mitochondrial protein concentration was 5.5 mg/ml for the spectra recorded from 490 to 630 m/r and 2.4 mg/ml for spectra recorded from 370 to 490 mr. DOC (240 PM) was added to the contents of the experimental cuvette, and the difference spectrum, Curve A (dashed line), was recorded. An equal concentration of DOC was then added to the reference cuvette in order to establish a baseline of equal light absorbance. Sodium malate (5 mM) was added to the contents of the experimental cuvette, and the reduction of pigments during the aerobic steady state, Curve B (dotted line), was recorded. After utilization of the oxygen in the experimental cuvette, Curve C (solid line) was recorded. The subsequent addition of Na&04 causes a further increase in absorbance (Curve D).
bands with maxima at 555 and 432 rnp. In this instance the absorbancy change associated with the absorption band of reduced cytochrome P-450 with its maximum at 432 rnp is considerably more positive relative to the baseline than seen in Fig. 3 (curve C), and supports the conclusion that the steroidinduced trough with a minimum at 420 ml remains even in the absence of oxygen. As described above for the experiments presented in Fig. 3, the subsequent addition of sodium dithionite causes a further reduction of the cytochrome, presumably resulting
from the loss of the spectral contributions associated with DOC interaction wit.h cytochrome P-450. Effect of oxygen on Doe-induced spectral changes. Experiments were then carried out to establish directly whether the DOC-induced spectral change remains in the absence of oxygen. As shown in Fig. 5, curve A, the typical decrease in absorbance was obtained when excess DOC was added to the experimental cuvette using mitochondria diluted in isotonic buffer in the absence of any substrates or inhibitors. Malate was then
742
CAMMER
AND
ESTABROOK
d A
_--- B
_.... .. .. C
\r’
A. DOC Minus Oxidized
I I
;: i’; :I i,
B. Malate Plus DOC (anaerobic)
Minus
Malate (anaerobic)
C. Succinate
Plus DOC (anaerobic) Succinate
Minus
(anaerobic)
i..: , I
‘1,’ -420 I 1 ' ' 1 I ' ' ' ' I 1 ' ' ' , ' ' 1 1 , 400 450 500 550 60( Wavelenqth (mp)
FIG. 5. Spectral changes associated with DOC interaction with cytochrome P-450 in the aerobic and anaerobic states. A 0.6.ml aliquot of beef adrenal cortex mitochondria (45 mg protein/ml) was diluted to 6 ml with buffer and divided equally into two cuvettes. DOC (240 PM) was added to the experimental cuvette, and the difference spectrum, Curve A (solid line), was recorded. Sodium malate (5 mM) was then added to the contents of both cuvettes, and after exhaustion of oxygen by the mitochondria in both cuvettes, Curve B (dashed line) was recorded. A second aliquot of mitochondria was diluted and treated in a similar manner except for the addition of succinate as substrate. The spectral change caused by the presence of DOC with succinate treated anerobic mitochondria is shown by Curve C (dotted line).
added to both cuvettes to initiate respiration, The appearance of an absorption band at 442 rnp indicated that oxygen had been exhausted first in the experimental cuvette containing DOC, followed by the subsequent disappearance of the absorption band of cytochrome oxidase at 442 rnp, showing that oxygen had been exhausted via the respiratory chain in the mitochondrial suspension present in the reference cuvette. The difference spectrum (curve B) was then recorded, showing directly the presence of the DOCinduced spectral trough in the anerobic sample. Trace C was bbt’ained in a similar manner, but succinate rather than malate was used as substrate. Clearly the absorption
decreases due to the presence of DOC remained with oxygen absent from the sample, although there were some displacements in the location of the minima. The difference in location of the absorption bands resulting from the presence of DOC in the aerobic or anerobic sample are similar to those described previously (35, 39) for substrate interaction of drugs with oxidized and reduced cytochrome P-450 of liver microsomes. When a similar series of experiments was carried out, using sodium dithionite as reductant, the spectral change associated with DOC interaction was obliterated, suggesting that chemical reduction with sodium dithionite, in contrast to enzymic reduction via the
SPECTRA
OF ADRENAL
cytochrome P-450 respiratory chain, markedly modified DOC interaction with cytochrome P-450. Spectral properties of the cytochrome P-450. CO complex. Cytochrome P-450 was first recognized by the large absorption band appearing at 450 mp after gassing a sample of the reduced pigment with carbon monoxide. Although cytochrome P-450 has been described as a component of microsomes from a number of different organs as well as a component of adrenal cortex mit’ochondria (cf. Fig. 2 and Refs. 2, 18, 19, 30), there have been no reports on associated spectral changes in the visible part of the spectrum. A series of spectral studies was therefore carried out with adrenal cortex mitochondria to establish t#he nature of t,he CO complex of cytochrome P-450 in this part of the spectrum. Fig. 6 (curve R) demonstrates that CO gassing of a sample of adrenal cortex mitochondria pretreated with succinate, cyanide, and DOC, followed by malate oxidation to establish anerobiosis, results in a displacement of the absorbance difference (cf. curve A) but no obvious format’ion of any new absorption bands. It is only when the spectral difference between the CO gassed anerobic sample and the malatetreated, non-CO gassed anerobic sample is examined t,hat the absorption properties of t,he CO complex of cyt,ochrome P-450 are detected. As shown in Fig. 6, curve C, rather broad absorption maxima at about 570 and 545 mp are observed. These spect)ral studies also revealed the presence of a weak absorption band with a maximum at about 647 rnp which disappears when the sample becomes anerobic during malate oxidation. This spectral change is associated with the appearance of t,he 555 m/l absorption maximum of reduced cytochrome P-450. Since cytochrome P-450 can be reduced eneymically either in the presence or absence of hydroxylatable substrate (DOC) and by addition of tricarboxylic arid cycle substrates such as succinate, malate, isocitrate, etc., spectral studies were carried out to determine whether there was any variation in the pattern of the spectral properties of the CO complex of reduced cyt,ochrome P-450. As shown in Fig. 7, the magnitude of the absorption bands with maxima at about 575
CORTEX
\ -006
743
MITOCHONURIA
/
i
I
B Malate (onoerobvz) + CO Minus Oxldlzed C Molate (anaerobic)+ CO Minus Malate kmaeroblc)
I”“i”“l”“I”l’
500
550 Wavelenqth
600
650
cm+)
FIG. 6. The effect of CO on the visible absorption of reduced cytochrome P-450. A 0.5.ml aliquot of adrenal cortex mitorhondria (44 mg/ml) R~S diluted to 6 ml wit,h isotonic bufier. Potassium cyanide (1 mM), sodium sllcciuate (5 mn~), and I>OC (0.24 mM) were added to the mixtrue, and the sample was divided eqllally ink) two cllvettes. After est,ablishment of a baseline of eqllal light absorption, sodium malate (5 mv) was added to one cuvette (experimental) aud the sample was permitted to ntilize the oxygen dissolved in the react.ion medium. The differenre spectrrrm of reduced cgtochrome P-450 (CIIIVC A) was then recorded. The contents of the anaerobic sample were theu gassed for 30 seconds with CO and the spectral changes recorded (Curve B). An aliqllot of sodirlm malate (5 mM) was t)heu added to the contents of the reference cllvette, alld, after attaining anaerobiosis, the difference spectrlml (Cltrve C) of the rcdrlced cytochrome P-45O’CO complex minrls reduced cytochrome P-450 was recorded.
and 545 rnp varied, depending on the conditions employed. Examination of t,he spectral region from 400 to 500 rnp revealed no discernible difference in the spectral properties of the cytochrome P-45O.CO complex. It appears that the presence of DOC favors the spectral changes associated with the formation of cytochrome P-450 ‘CO complex at 575 rnp, and the absence of hydroxylatable substrate favors the formation of a compound with a spectral maximum at about 545 rnp. These differences may be correlated with the possible change in the strength of ligand binding occurring under t.he various conditions examined. Correlation to the spect:ral properties of cytochrome P-450 of liver microsomes. Previous reports (18, 42) have demonstrat’ed that
744
CAMMER AND ESTABROOK
A Swcwwie(Anambic~+ CO MINIS SuccinotdAnaerobic) E Succ~nots(Amembic)+DOC+CO Mmur S~~~c~nols(Ar.wcbiil+DOC C Succimfe+KCN+Moi~klA~robicl+CO Minus Succirmie+KCN+M~late(Anoerobicl I 490
I I I I r I, 520 550 560 Wovelength
I, 610
I I ,I 640
FIG. 7. The spectral properties of the reduced cytochrome P-45O.CO complex obtained with varying substrates in the presence and absence of DOC. Experiments were carried out as described in Fig. 6. For Curve A, the diluted adrenal cortex mitochondria in the absence of DOC and cyanide were keated with sodium sucoinate (5 mM) and the anerobic sample in the reference cuvette was gassed for 30 seconds with CO. The difference spectrum presented in Curve B was obtained with diluted adrenal cortex mitochondria, to which DOC (0.24 mM) and sodium succinate (5 mM) were added prior to gassing with CO. The difference spectrum of reduced cytochrome P-45O.CO complex observed in the presence of potassium cyanide (1 mM), sodium succinate (5 mM), and sodium malate (5 mM) in the absence of DOC is shown in Curve C.
sodium dithionite reduces a pigment of liver microsomes not reduced during the aerobic steady state by DPNH. Mason et al. (20) have also demonstrated the enzymic reduction of a similar pigment by TPNH under anerobic conditions. To determine whether these spectral changes may represent the spectral contribution of reduced cytochrome P-450, experiments of the type presented in Fig. 8 were carried out. For these experiments microsomes were obtained from livers of rabbits treated with phenobarbital (43) to cause maximal spectral change on reduction of cytochrome P-450. Curve A of Fig. 8 illustrates the spectral contribution of cytochrome bgof liver microsomes reduced during the aerobic steady state by TPNH. No significant difference was observed whether TPNH or DPNH was used as reductant. The subsequent addition of N&z04 to the sample (curve B) caused a pronounced increase in absorbance at about 556 rnp, to-
gether with the appearance of a trough at about 576 mp. When the difference spectrum is measured between a sample treated with Na&04 to reduce both cytochrome bs and cytochrome P-450 and a sample treated aerobically with TPNH or DPNH, the difference spectrum of reduced minus oxidized cytochrome P-450 (curve C) is obtained. A similar type of spectral change can also be obtained when TPNH rather than NazSz04 is added to an anerobic sample, and confirms the earlier observation of Mason et al. (20) and the recent report by Nishibayashi and Sato (44). The shape of the spectral change associated with reduction of cytochrome P-450 of rabbit liver microsomes is remarkably similar to that observed upon reduction of cytochrome P-450 of adrenal cortex mitochondria (cf. Figs. 1, 3, 4, and 6). The subsequent addition of CO to the anerobic sample of rabbit liver microsomes results in the formation of two rather weak absorption bands with maxima at about 545 and 575 rnp comparable with that described in Fig. 6 for the spectral contribution of cytochrome P-450. CO complex of adrenal cortex mitochondria. Extinction coeJ.kients of cytochrome P-&O. The experiments described above now permit the identification of spectral changes, as observed by difference spectrophotometry, associated with cytochrome P-450. The millimolar extinction coefficient of the reduced cytochome P-450. CO complex with a maximum at 450 rnp has been determined by Omura and Sato (19) to be 91 rnr’ cm-‘. This extinction coefficient was used as the basis for determining the content of cytochrome P-450 in a sample, and the change in extinction coefficients occurring upon reduction of cytochrome P-450 and the complexing of reduced cytochrome P-450 with CO was determined (Table 11). Of interest is the relatively low extinction change occurring at 432 rnp on reduction of cytochrome P-450. The very low extinction change observed in the visible portion of the spectrum upon formation of the CO complex of reduced cytochrome P-450 explains the failure to detect these absorption bands in previous experiments. A comparison~_of the _ changes in extinction observed with adrenal cortex
SPECTRA TABLE
OF AURENAL
II
EXTINCTION CH.LNGES OCCURRINO DURING CYTOCHROME P-450 REDUCTIONOR~NTER.4CTION WITH SURSTRaTE" Condition
\Vavt>h
A(m1.0 cm-l)
432-490 555-576 420-490 573-600 450-490 570-59
24 10 30 4 91 2.5
Anerobic minns aerobic Aerobic + DOC mintis aerobic Anerobic + CO minus anerobic
a Experiments were carried out as described in Figs. 3-6. In each experiment the content of cytochrome P-450 was determined from the absorbance change at 450 rnr relative to 490 rnp observed after rediiction with Na&04 and gassing with CO, employing the extinction coefficient recommended by Omura and Sato (19). Comparable results have beeu obt)ained with microsomes from rabbit liver.
CORTEX
715
MITOCHONDRIA
certain whether cytochrome P-450 may exist in two forms in adrenal cortex mitochondria or liver microsomes (38). One form predominates in the presence of a hydroxylatable substrate, such as DOC, while the other occurs in the absence of substrate. The difference between the two forms may reflect an alteration in Iigand binding to the heme of cytochrome P-450 as described previously (38, 39, 41), or it may result from monomer to dimer transitions of the hemoprotein. These two forms are observed by the spectral changes occurring in the presence and absence of DOC during the experiments with adrenal mitochondria. In many instances these differences are smal1 and difficult to detect, but in other instances, such as the 555
I
/
1
;: ,!
mitochondria and those observed with microsomes from rabbit liver indicates a very close correlation for the two types of systems. DISCUSSION
The present studies have attempted to define the spectral changes occurring upon reduction of pigments associated with substrate oxidation by beef adrenal cortex mitochondria. These mitochondria are of interest be&use they contain not only the “normal” respiratory chain of mitochondria functional in the synthesis of ATP but also a second type of respiratory chain containing cytochrome P-450 functional in the hydroxylation of DOC and cholesterol. Quantitatively, adrenal cortex mitochondria contain a complement of cytochromes a + u3, b, and c + cl very similar to that reported for liver mitochondria. As reported by Harding and Nelson (2), the preponderant pigment of these mitochondria is cytochrome P-450. Utilizing inhibitors of the normal respiratory chain, it was possible to assess the spectral changes occurring during reduction of cytochrome P-450 and its interaction with DOC. From these studies the tentative scheme presented in Fig. 9 was constructed. The wavelengths designated indicate the absorption maxima or minima observed upon transition from one state to another. It is un-
I
I
490
I
I
I
520
r
8,
/
550
I,
II,
580
I
610
I
I
640
I
/
,
660
Wavelength(mp1
FIG. 8. The difference spectrum of reduced cytochrome P-450 of rabbit liver micrdsomes. A 0.6.ml aliquot (30 mg protein/ml) of mi&osomes prepared from livers of rabbits pretreated for 5 days by daily injections @Omg/kg) of phenobarbital was diluted to 6 ml with 50 mM Tris buffer, pH 7.4. The contents of the diluted sample were divided equally into two cuvettes and a baseline of equal light absorption was recorded. A 0.2.ml aliquot of a TPNII solution (20 mM) was added to the contents of the experimental cuvette, and the difference spectrum of reduced cytochrome bs (Curve A) obtained during the aerobic steady state was recorded. A few crystals of solid sodium dithionite were then added to the contents of the same cuvette, and the difference spectrum (Ciirve B) was recorded. After addition of a 0.02-11~1aliquot of a TPNH solution (20 mM) to the contents of the reference cuvette to reduce cytochrome b,, the difference spectrum (Curve C) of redrlced minus oxidized cytochrome P-450 was recorded.
746
CAMMER
AND
ESTABROOK
P-450 Fe”,DOC Cb
Co$os$rone
2 FIG. 9. Schematic representation of possible oxidation and cytochrome P-450 in the presence or absence of deoxycorticosterone
visible spectral changes occurring upon formation of the CO complex of reduced cytochrome P-450, the spectral properties of the pigment can be markedly modified. The ability to now measure the spectral changes occurring upon reduction of cytochrome P-450 permits experiments to evaluate directly the interaction of oxygen with this pigment during hydroxylation reactions. It is apparent from the studies presented here that cytochrome P-450 is largely in the oxidized form during the aerobic steady state of hydroxylation when adrenal cortex mitochondria or liver microsomes are employed. The kinetic transitions occurring during the oxidation and reduction of cytochrome P-450 will be described in a subsequent paper. In the present study, no evidence was found which could argue in favor of or against an oxygenated form of ferrous cytochrome P450. Also, since the present studies employed turbid suspensions of mitochondria or microsomes and therefore have been restricted to spectral changes assessed only by difference spectrophotometry, it has not been possible
reduction changes of (DOG), CO, and OZ.
to assign absorption maxima to the ferric form of the hemoprotein. However, it has been possible to determine quantitatively the cytochrome content of adrenal cortex mitochondria and to assign changes in extinction occurring during the oxidation and reduction of these pigments. REFERENCES 1. CAMMER, W., AND ESTABROOK, R. W., Arch. Biochem. Biophys. 122, 721-734 (1967). 2. HARDING, B. W., AND NELSON, D. H., J. Biol. Chem. 241, 2212 (1966). 3. PURVIS, J., BATTU, R., AND PERON, F. G., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 4. KORITZ, S. B., Biochem. Biophys. Res. Commun. 23, 485 (1966). 5. MCCARTHY, J. L., AND PERON, F. G., Biochemistry 6, 25 (1967). 6. NAKAMURA, Y., OTSUKA, H., AND TAMAOKI, B. I., Biochim. Biophys. Ada 122, 34 (1966). 7. SWEAT, M. L., J. Am. Chem. Sot. 73, 4056 (1951). 8. HAYANO, M., AND DORFMAN, R. I., J. Biol. Chem. 211, 227 (1954).
SPECTRB
OF ADRENAL
9. Ha~axo,hl., LINUBERG, &I. C., DORFMSN, R. I., HANCOCK, J. E. H., .\XD DOEKING, W. VON E., .4rch,. Biochem. Riophys. 59, 529 (1955). 10. ESTABROOK, R. W., COOPER, 11. Y., AND ROSENTHAL, O., Hiochem. Z. 383,741 (1963). 11. COOPER, 1). Y., LEVIN, S., NARASIMHULU, S., ROSENTHAL, O., AND EST.~BROOK, R. W., Science 147, 400 (1965). 12. H.\RDING, B. W., WONG, S. H., AND NELSON, D. II., B&him. Biophys. Beta 92, 415 (1964). 13. WILS~X,L. D.,NELsoN,D. H., AND HARDING, B. W., Biochim. Biophys. ;lcta 99,391 (1965). 14. COOPER, I). Y., NAR.~SIMHULU, S., HL.~DE, A., XIICH, W., FOHOFF,~., ANT) ROSENTH~L,~., Life 67-i. 4, 2109 (1965j. 15. CAMMER, W., COOPER, D.Y., AND EST.~BROOK, 1~. W., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (19G7). 16. HARDING, B. W., OLUHAM,S. B., ANU WILSON, L. I>., in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 17. SIMPSON, E. It., .\ND BOYD, G. S., Biochem. Biophys. Res. Commun. 24,lO (1966). 18. COOPER, D. Y., NIRASIMHULU, S., ROSENTHAL, O., AND EST;ZBROOK, R. W., in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 841, Wiley, New York (1965). T., .~PI‘DSATO, R., J. Biol. Chem. 233, 19. OMURA, 2270 (1964).20. MASON, H. S., NORTH, J. C., AND VANNESTE, M., Federation Proc. 24,1172 (1965). 21. SPIRO, M. J., .~ND BALL, E. G., J. Biol. Chem. 236, 231 (1961). 22. RINOSHITB, T., HORIE, S., SHIMAZONO, N., .~ND YOHRO, T., J. Biochem. (Tokyo) 60, 39 (1966). 23. HoRIE,S.,KINOSHITA,T.,.~NDSHIMAZONO,N., .I. Biochem. (Tokyo) 60,660 (1966). 24. KI~~uR.?, T., in “Biological and Chemical Aspects of Oxygenases” (K. Bloch and 0. Hayaishi, eds.), p. 179. Maruzen Co. Ltd., Tokyo (1966). 25. CHANCE, B., Methods Enzymol. 4,273 (1957). 2fj. CORNALL, A. G., BARDAWILL, C. J., AND DAVID, h1. M., J. Biol. Chem. 177,751 (1949). 27. CAMMER, W., AND ESTABREOK, It. W., Federation Proc. 25, 281 (1966). 28. GRANT, J. K., .\ND BROWNIE, A. C., B&him. Biophys. Acta 18, 433 (1955). 29. S~~EAT, 1LI. L., ANI) LIPSCOMB, M. I~., J. Am. Chem. Sot. 77, 5185 (1955). 30. OMURA,T., s.4~0, R., COOPER,D.Y.,ROSENTHBL, O., AND ESTABROOK, R. W., Federation Proc. 24, 1181 (1965).
CORTEX
MITOCHONl)l~IA
747
31. EST.~IIROOK, R. W., SCHENKMIN, J. B., CAMMER, W., REMMER, H., COOPER, 1). Y., N.\R,~SIMHULU, S., .\ND ROSENTH.~L, O., in “Biological and Chemical Aspects of Oxygenases” (K. Bloch and 0. Hayaishi, eds.), p. 153. Maruzen Co. Ltd., Tokyo (1966). 32. ESTABRO~K, 1~. W., .IND HOLO~INSKY, A., J. Biophys. Biochem. Cytol. 9, 19 (1961). 33. ESTBBROOK, R. W., AND M.~ITR.~, P. K., dnd. Biochem. 3, 369 (1962). T., SANDERS, E., COOPER, I). Y., 34. OMURA, ROSENTHAL, O., ABD Es~an~ooK, 1~. W., in “Non-Heme Iron Proteins: Role in Energy Conservation” (A. San Pietro, ed.), p. 401; Kettering Symposilun. Antioch Press, Yellow Springs, Ohio (1905). 35. CMURA, T., CAMMER, w., COOPER, D. Y., ROSENTHAL, O., AND ESTABROOK, It. W., in “Elektrochemische Methoden und Prinzipien in der Molkealar-Biologie” (H. Berg, ed.), p. 25; Third Jaener Symposium. Akademie-Verlag, Berlin (1966). 36. KLINGENBERG, M., in “Fnnktionelle und Morphologische Organisat,ion der Zelle” (P, Karlson, ed.), p. 69. Springer-Verlag, Berlin (1963). 37. NARASIMHULU,S.,COOPER, D.Y., ANDROSENTHAL, O., Life Sci. 4, 2101 (1965). 38. COOPER, D. Y., NIRASIMHULU, S., ROSENTHAL,~., AND EsT.~BROOK, R. W.,in “Symposium on the Functions of the Adrenal Cortex” (K. W. McKerns, ed.). Appleton, New York (1967). 39. REMMER, H., &HENKMSN, J. B., ESTABROOK, R. W., SASAME, H., GILLETTE, J., NARASIMHULU,S.,COOPEK, D.Y., AND ROSENTHAL, O., Mol. Pharmacol. 2, 187 (1966). 40. IMAI, Y., AND SATO, li., Hiochem. Biophys. Res. Commun. 23, 5 (1966). 41. ESTABROOK, 13. W., PCHENKMAN, J. B., CurMER, W., COOPER, D. Y.,NARASIMHULU, S., S., .\NI) ROSENTH.\L, O., in “Hemes and Hemoproteins,” (B. Chance, R. W. Estabrook, and T. Yonetani, eds.), p. 511. Academic Press, New York (1967). 42. S.4~0, R., OMURA, T., .\ND NISHIBAYASHI, H., in “Oxidases and Related Redox Systems” (T. E. King, 1-I. Mason, and M. Morrison, eds.), p. 861. Wiley, New York (19G5). 43. REMMER, H., GREIM, II., SCHENKMAK, J. B., AND ESTABROOK, R. W., in “Methods in Enzymology,” Vol. 10 (“Oxidation and Phosphorylat,ion”; R. W. Estabrook and M. PulIman, eds.), p. 703. Academic Press, New York (1967). 44. NISHIBAYASHI, H., AND 89~o,R., J. Biochem. 61, 491 (1967).