170

Biochimica et Biophysica Acta, 577 (1979) 170--176

© Elsevier/North-Holland Biomedical Press

BBA 38132 E L E C T R O N PARAMAGNETIC RESONANCE STUDIES OF THE P U R I F I E D CYTOCHROME P-450s¢~ AND P-45011~ F R O M BOVINE A D R E N O C O R T I C A L MITOCHONDRIA

SHIRO KOMINAMI, HIROSHI OCHI and SHIGEKI TAKEMORI Department of Environmental Sciences, Faculty of Integrated Arts and Sciences, Hiroshirna University, Hiroshima 730 (Japan)

(Received July 19th, 1978) Key words: Cytochrome P-450; ESR; High spin heme; Low spin heine; (Adrenal mitoehondria)

Summary Electron paramagnetic resonance studies have been carried o u t on t w o species of c y t o c h r o m e P-450 (P-450sc¢ and P-4501~) purified from bovine adrenocortical mitochondria. The g values of the steroid-bound cytochromes in the high spin form were determined at 4.2 ° K to be 8.07, 3.60 and 1.70 for P-450sc¢ and 8.00, 3.65 and 1.71 for P - 4 5 0 ~ . The E / D values were estimated to be 0.103 for P-450,cc and 0.099 for P - 4 5 0 ~ . Either high spin P-450 was convetted into the low spin form by the treatment with an NADPH dependent electron donating system and subsequent gel filtration in order to remove the steroid. The g values of the low spin ferric cytochromes were 2.423, 2.247 and 1.914 for P-450,cc and 2.430, 2.251 and 1.919 for P - 4 5 0 ~ at 77 ° K. The values for iA/kl, iV/kf and k were 5.69, 5.21 and 1.11 for P-450sc c and 5.94, 5.38 and 1.16 for P - 4 5 0 ~ . These studies indicate that there are some differences in the ferric heme environment between P-450,¢c and P-45011~.

Introduction T w o different kinds of c y t o c h r o m e P-450 (henceforth called P-450) are required for the steroid hydroxylation in adrenal mitochondria. One is P-450sc c which is responsible for the side chain cleavage of cholesterol and the other is P-4501~ that catalyzes 11~- as well as 18.hydroxylation of deoxycorticosterone. Several electron paramagnetic resonance (EPR) studies have been carried o u t on the isolated adrenal mitochondria and the partially purified P-450 preparations [ 1--9]. From these studies it has b e c o m e apparent that EPR

171 signals of P-450,~ differ somewhat from those of P - 4 5 0 ~ in both a high spin and a low spin state [5--8]. It has been suggested that a considerable amount of P-450scc in intact mitochondria is complexed with the endogenous cholesterol since P-450scc is predominantly in a high spin state [7--9]. The spin state conversion of P-450 by substrate binding has been clearly demonstrated in P'450cam from Pseudomonas putida [10]. In order to provide the direct and most convincing evidence regarding the spin state of adrenal P-450, it is important that both P-450~c~ and P-450~,~ are available in pure form. Recently, each P-450 has been highly purified in a functionally active form from bovine adrenal mitochondria [11--16]. The preparation is free from the other in respect to the enzyme activity and electrophoretic and immunological properties. This obviously makes it possible to compare physical characteristics between the two cytochromes in detail. The purpose of the present investigation is to examine the EPR properties of both a high spin and a low spin form of P-450s¢¢ and P-450, ~ purified from bovine adrenal mitochondria. Materials and Methods

Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, dithiothreitol and NADP ÷ were obtained from Boehringer und Sohme, GmbH, Mannheim. 20aHydroxycholesterol was from Sigma Chemical Co., St. Louis. Deoxycorticosterone and pregnenolone were from Nakarai Chemicals Ltd., Kyoto. All other chemicals were of the highest commercially available grades. The buffer used was 50 mM Tris-Cl (pH 7.3) containing 100 ~M EDTA and 100 uM dithiothreitol. Adrenal ferredoxin and NADPH-adrenal ferredoxin reductase were prepared as described previously [17,18]. P-450~cc and P-45011~ were purified from bovine adrenal mitochondria according to the method of Takemori et al. [11,12] as modified by Suham et al. [14]. The heme content was 11 to 12 nmol/mg protein for the purified P-450,1~ and P-450~cc. The purified P-450~¢c contained endogenous cholesterol (about 1.0 mol]mol of heme). The P-45011~ contained deoxycorticosterone which had been added as a stabilizing agent during purification and storage. The P-450~c~ was finally dialyzed against the buffer containing 0.01% sodium cholate. The P-45011~ was dialyzed against the buffer containing 10 ~M deoxycorticosterone, 0.3% sodium cholate and 0.3% Tween 20. The sample of a high spin form was concentrated into 0.2 mM using a macrosolute concentrator (Minicon B 15) and was filled in a 1 mm quartz EPR sample tube. The tube was immediately frozen in liquid nitrogen. In order to convert a high spin into a low spin form, the high spin P-450 (20 nmol} was incubated with the electron donating system consisting of 9.7 nmol of adrenal ferredoxin, 0.56 unit of NADPH-adrenal ferredoxin reductase, 0.56 ~mol of MgC12, 20 ~mol of glucose 6-phosphate, 0.65 unit of giucose-6-phosphate dehydrogenase and 13 nmol of NADP ÷ in the buffer (2.4 ml). After the reaction was completed, the solution was passed through a Sephadex G-25 column (1 X 10 cm) previously equilibrated with the buffer containing 0.3% Tween 20 in order to remove the steroid product and immediately concentrated into about 0.2 mM. The low spin P-450 did not contain any detectable amount of steroid. The low spin sample was filled in a 3 mm quartz EPR sam-

172 ple tube. The inactive form (P-420) was prepared by the treatment of the P-450 preparation with 0.75 M KSCN [19]. The EPR measurements were performed with JEOL-ME-3X operated in the x-band with 100 KHz field modulation. The strength of the magnetic field was determined with a proton resonance of H20 using a Robinson-type oscillator and a frequency counter, TR-5578, of Takeda Riken. The frequency of the microwave was determined using a,a'-diphenyl~-picrylhydrazyl radical [ 20]. Results and Discussion Fig. 1 shows the first derivative EPR spectra of the purified P-450scc and P-45011~ which have optical absorption maxima at 394, 510 and 645 nm, characteristic of a high spin ferric heme. The signals around g = 8, 3.6 and 1.7 are due to a high spin ferric heme and those from 2500 G to 3500 G can be attributed to a low spin ferric heme. The observed g values of the high spin P-450 are listed in Table I. These g values were quite similar to those observed for P-450ca m [10] and P-450 from rat liver microsomes [21]. The low field peak for the high spin P-450scc was at the lower magnetic field than that for the high spin P-4501~ and the center of the middle field signal of the P-450s~c was at the higher magnetic field than that of the P-450~z. The peak widths were different between those signals. The width at the half-height of the low field peak was 22 G for the P-450scc and that for the P-450~1~ was 29 G. T h e peak to peak widths for the middle field signals were a b o u t 130 G and 160 G for the P-450scc and the P-4501~, respectively. The difference in the line width may be due to the difference in the spin-lattice relaxation time (T1). The low spin signals in the P-450scc were quite distorted at 4.2 ° K and this is probably due to the microwave power saturation. Three low spin signals in the high spin P - 4 5 0 ~ were apparent and these were not observable at 77 ° K. All of the other low spin signals observed in this experiment were distorted at 4 . 2 ° K and

(b)

w

....

eoo ~eo

2ooo I

o.e.o g~.o

I

g-~so

i

f

-

3ooo I

-

4opo~s~.,~

I

l

g'2.o

i

o-,.7 •

Fig. 1. T h e first derivative EPR s p e c t r a o f high spin ferric c y t o c h r o m e P~450sc c (a) and P-45011~ (b) puxifled from bovine adrenocortical mitochondria. S p e c t r a w e r e o b t a i n e d u n d e r t h e f o l l o w i n g c o n d i t i o n s : m i c r o w a v e power, 3 mW; field m o d u l a t i o n amplitude, 5 G; field s c a n s p e e d , 5 0 0 G/rain; t e m p e r a t u r e , 4.2* K.

173 TABLE I T H E E P R P A R A M E T E R S O F T H E H I G H SPIN F E R R I C C Y T O C H R O M E P - 4 5 0 Samples

Observed g values

C o m p u t e d g values

E/D v a l u e s

P-450sc c

8 . 0 7 2 0.01 3 . 6 0 +_ 0 , 0 2 1.70 ± 0,02

8.08 3.59 1.70

0.103

P-45011~3

8 . 0 0 ± 0.01 3.65 + 0 . 0 2 1.71 + 0 . 0 2

8.02 3.66 1.71

0.099

apparent at 77 ° K. The g values of the low spin signals in the high spin P-450~t~ were 2.397, 2.236 and 1.974 which were different from those of the low spin substrate-free form or from those of P-420. The g values of a high spin ferric heme are well described by the term of E/D, where E and D represent the tetragonal and rhombic distortions of the crystal field from the octahedral symmetry [22]. The E/D values in Table I were determined by fitting the observed to the calculated g values by using the same approximation as described by Sato and Kon [23]. The calculated g values agree fairly well with the observed values. The percentage of the rhombicity, which is expressed as E / D × 3 × 100% [24], is 31% for the P-450scc and 27% for the P-4501~. In order to estimate the amount of the low spin portion in the high spin sample, the absorption area of its low spin spectra produced by treating with the NADPH dependent electron donating system or with KSCN as described below, was compared with that of the low spin absorption in the high spin samples. The amount of low spin form was about 5 to 10% of the total EPR absorption in the high spin samples of P'450see and P-4501~. This value is significantly

(o) -

+

'

~

~

(b)

2600

3000

I

I

3400 (Gouss) I e

Fig. 2. T h e first d e r i v a t i v e EPR spectra o f l o w spin ferric c y t o c h r o m e P - 4 5 0 s c c (a) a n d P - 4 5 0 1 1 ~ (b) p r o d u c e d b y t r e a t m e n t w i t h the N A D P H d e p e n d e n t e l e c t r o n d o n a t i n g s y s t e m . S p e c t r a w e r e o b t a i n e d u n d e r t h e f o l l o w i n g c o n d i t i o n s : m i c r o w a v e p o w e r , 8 m W ; field m o d u l a t i o n a m p l i t u d e , 10 G ; field scan s p e e d , 1 0 0 G / m i n ; t e m p e r a t u r e , 77 e K.

174 smaller than those reported for P'450eam from Pseudomonas putida [10] and P-450 from rat liver microsomes [21]. When the high spin P-450 was treated with the NADPH dependent electron donating system and subsequently applied to gel filtration in order to remove the steroid product, the prepared P-450 was characteristic of the low spin form having the optical absorption maxima at 418, 539 and 570 nm. The first derivative EPR spectra of the low spin P-450 recorded at 77 ° K are shown in Fig. 2. When the low spin sample was measured at 4.2°K, a small signal around g = 8 and a large distorted signal from 2500 G to 3500 G were observed, suggesting that the conversion to the low spin form was n o t absolutely complete. The pattern of saturation with microwave power of the low spin signals was determined and the spectra of the low spin samples were recorded under nonsaturating conditions at 77 ° K. In the spectrum of the low spin P-450sce, there was a small shoulder at g = 2.45 and a broad signal was superimposed on the middle signal. These small signals could n o t be eliminated in several series of experiments. These might be artificially altered forms such as P-420 contaminated in the sample. The g values of the low spin signals were listed in Table II. The high spin P'450sec was also converted into the low spin form upon the addition of pregnenolone or 20~-hydroxycholesterol. The g values of the low spin signals of P-450scc produced b y these steroids were a little different from those of the substrate-free P'450sec. The spin state conversion has been reported to occur with the conversion of the high spin P-450 to the P-420 [25]. The EPR spectra of the P-420 derived from P-450se¢ and P-4501~ were n o t so much different from those of the low spin P-450. It is of some interest that the g values of these P-420 differ slightly from each other. For the low spin ferric heme, the g factor has been discussed b y Griffith [26,27]. The anisotropy in g factor is a function of A/X and V/k, where A and V represent the tetragonal and the rhombic distortions from the octahedral crystal field, respectively and X is the spin-orbital coupling constant. The values for A/X and V/X can be estimated from the observed g values with the orbital reduction factor k. There are 48 possible combinations depending on the labelling (x, y, z) and the signs chosen for the observed g values. In the several T A B L E II T H E E P R P A R A M E T E R S O F T H E L O W SPIN F E R R I C C Y T O C H R O M E P - 4 5 0 D E R I V A T I V E S Samples

L o w spin P - 4 5 0 s c c

O b s e r v e d g values

C r y s t a l field p a r a m e t e r s

k

I~/xl

Iv/xl

2 . 4 2 3 + 0 . 0 0 3 , 2 . 2 4 7 _+ 0 . 0 0 2 , 1 . 9 1 4 -+ 0 . 0 0 4

1.11

5.69

5.21

2 . 4 0 3 _+ 0 . 0 0 2 , 2 . 2 4 2 + 0 . 0 0 2 , 1.921 +- 0 . 0 0 1

1.11

5.79

5.47

2 . 4 3 7 +_ 0 . 0 0 3 , 2 . 2 4 5 + 0 , 0 0 3 , 1 . 9 1 2 +- 0 . 0 0 2

1.12

5.85

5.09

(steroid free) High spin P - 4 5 0 s c c + 20c~-hydroxyeholesterol High spin P - 4 5 0 s c c +

pregnenolone P-420 derived from P-450sc c

2 . 4 8 7 _+ 0 . 0 0 6 , 2 . 2 7 4 + 0 . 0 0 3 , 1 . 8 9 4 + 0 . 0 0 7

1.15

5.33

4.68

L o w spin P - 4 5 0 1 1 ~ (steroid free) P-420 derived from P-45011 ~

2 . 4 3 0 +_ 0 . 0 0 2 , 2 . 2 5 1 + 0 . 0 0 2 , 1 . 9 1 9 + 0 . 0 0 2

1.16

5.94

5.38

2 , 4 6 0 + 0 . 0 0 4 , 2 . 2 7 2 +_ 0 . 0 0 1 , 1 . 9 0 7 + 0 . 0 0 2

1.17

5.49

5.07

175 single crystal studies on the low spin ferric heme, the maximum g values have been observed at the orientation of the magnetic field along the z axis normal to the heme plane [28,29]. It is assumed in this study that the direction of the maximum g value should be along the z axis and the value for k should not be far from unity. The values for A/k, V/k and k obtained under such assumptions are listed in Table II. As can be seen, the values are quite similar for various low spin species. When these values were plotted on the crystal field diagram having the A/k as x axis and V/A as y axis, as described by Blumberg and Peisach [30--32], they fall within the same region as low spin ferric compounds having mercaptide sulfur and imidazole nitrogen for the axial ligands, even for the P-420. The value of the VIA is a measure of the rhombicity of a low spin ferric heme. It is of interest to notice that both high and low spin forms of P-450see show higher rhombicity than those of P-4501~. In conclusion, the results presented here show that there are some differences in the environment around the heme moiety between P'450sce and P-450~ 1~ and that the conversion from a high to a low spin state corresponds to the interaction of P-450 with the steroid substrate. Acknowledgements The authors gratefully acknowledge the technical advice of Dr. T. Izuka, Keio University School of Medicine, for making the liquid helium dewar and of Dr. K. Morimoto, Hiroshima University, for making the Robinson-type oscillator. This investigation has been supported in part by the research grant for 1976 from Matsunaga Science Foundation and Scientific Research Fund of the Ministry of Education of Japan (1976-1977, Grants 147132 and 278066).

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