252
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[26]
[26] D e t e c t i o n a n d Q u a n t i t a t i o n o f F r e e C y t o c h r o m e P-450 and Cytochrome P-450 Complexes by EPR Spectroscopy By NANETTE R. ORME-JOHNSON and W. H. ORME-JOHNSON
Cytochromes P-450 in the oxidized (Fe 3÷) state exhibit electron paramagnetic resonance (EPR) spectra at low temperature, both in the free form and when bound to substrates, some intermediates and products, or certain inhibitors. The EPR 1"2 spectra fall into two categories, those arising from a high-spin heme species (S = 5/2) with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. Figure 1 shows high-spin and low-spin signals from P-450eam. 3 In general, free (uncomplexed) cytochrome P-450 is the low-spin state. 4 This is true for example of P-450cam from P s e u d o m o n a s p u t i d a , 3 the P-450s from liver microsomes, 5 the P450 in placental microsomes, e and the P-450 (P-45011~) from adrenal mitochondria involved in the conversion of deoxycorticosterone to corticosterone. 7 In general, the binding of amines or other nitrogen containing ligands to P-450's produces low-spin species while the binding of subtrates (or some inhibitors) produces high-spin species, s A 1 G. Palmer, this series, Vol. 10, p. 598. 2 j. A. Fee, this series, Vol. 49 [20]. 3 R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Prec. Natl. Acad. Sci. U.S,A. 66, 1157 (1970). * An apparent exception to this is the P-450 (P-450~c) isolated from adrenal mitochondria and involved in the conversion of cholesterol to pregnenolone. This cytochrome is predominantly high spin as isolated; however, the isolated preparations contain at least a stoichiometric amount of cholesterol so that this high-spin species may be the P-450~ccholesterol complex. 5 A. Y. H. Yu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 44, 3714 (1969). e j. A. Canick, K. J. Ryan, N. R. Orme-Johnson, W. H. Orme-Johnson, and A. C. Brownie, Meeting of the Endocrine Society, 58th, 1976, San Francisco, Calif., p. 113. 7 C. R. Jefcoate, R. Hume, and G. S. Boyd, FEBS Lett. 9, 41 (1970). s Even when an excess of a ligand is present, there may be an equilibrium between the high-spin and low-spin forms so that measuring the amount of P-450 in the predominant spin form will not yield the total amount. However, in the cases for which this phenomenon has been quantitated, the amount in the alternate spin state is less than 10% of the total amount. Therefore, quantitation of the predominant form yields numbers that are very close indeed to the true value, and, quantitation of both spin forms gives the true value [S. G. Sligar, Biochemistry 15, 5399 (1976)].
[26]
253
DETECTION AND QUANTITATION OF CYTOCHROME P-450
79* K 245
15 ° K tge
I
2~15226 1~t911/~
450
PHE-
.IM ) y_)/ f~S----
P450 ÷ CAMPHOR W
FIG. 1. Electron paramagnetic spectra of Pseudomonas putida P-450 at 79 ° and 15°K. Top: 1.1 rnM P-450 untreated; center: after addition of 0.25 volume of a 6 mM solution of 1phenylimidazole; bottom: after addition of 0.25 volume of a 6 mM solution of D-(+)camphor. The conditions of EPR spectroscopy were at 79 ° and 15°K, respectively: microwave power, 27 and 0.27 mW; scanning rate 160 and 625 G/min; modulation amplitude 6 G and time constant 0.25 sec throughout. The amplification of the center and bottom curves, recorded at 79°K, is 1.25 times that of the top curve to correct for dilution and the amplification of the bottom curve recorded at 15°K is 3.1 times that of the others. From R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Pro('. Natl. Acad. Sci. U.S.A. 66, 1157 (1970).
few specific examples may hint at the enormous amount of information available from EPR spectra. P-450scc presents a more complicated picture when bound to substrate, postulated reaction intermediates, and product. P-450~c bound to cholesterol is high spin; bound to 20-ahydroxycholesterol or 22-R-hydroxycholesterol is low spin; bound to 20,22-dihydroxycholesterol is high spin; and bound to pregnenolone is low spin. Further it is possible to determine from the EPR spectra which of the steroids producing a low-spin complex is bound. 9 Similarly, high9 N. R. Orme-Johnson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1246, Abstr. 124 (1974).
254
MICROSOMAL ELECTRON TRANSPORTAND CYT P-450
[26]
spin P-450 complexes do not all have the same g values. For example in rat adrenal mitochondria, the P-450~l,.deoxycorticosterone complex has gmnx = 7.9 while the P-450~e'cholesterol complex has gmax = 8.1, so that it is possible to monitor these two species separately in the intact organelles,l° though the spectra are overlapped and may require spectral simulation for satisfactory quantitation.
Low-Spin Heine---Detection and Quantitation EPR spectra of low-spin cytochrome P-450 are easily observed at temperatures
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[26]
[26] D e t e c t i o n a n d Q u a n t i t a t i o n o f F r e e C y t o c h r o m e P-450 and Cytochrome P-450 Complexes by EPR Spectroscopy By NANETTE R. ORME-JOHNSON and W. H. ORME-JOHNSON
Cytochromes P-450 in the oxidized (Fe 3÷) state exhibit electron paramagnetic resonance (EPR) spectra at low temperature, both in the free form and when bound to substrates, some intermediates and products, or certain inhibitors. The EPR 1"2 spectra fall into two categories, those arising from a high-spin heme species (S = 5/2) with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. Figure 1 shows high-spin and low-spin signals from P-450eam. 3 In general, free (uncomplexed) cytochrome P-450 is the low-spin state. 4 This is true for example of P-450cam from P s e u d o m o n a s p u t i d a , 3 the P-450s from liver microsomes, 5 the P450 in placental microsomes, e and the P-450 (P-45011~) from adrenal mitochondria involved in the conversion of deoxycorticosterone to corticosterone. 7 In general, the binding of amines or other nitrogen containing ligands to P-450's produces low-spin species while the binding of subtrates (or some inhibitors) produces high-spin species, s A 1 G. Palmer, this series, Vol. 10, p. 598. 2 j. A. Fee, this series, Vol. 49 [20]. 3 R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Prec. Natl. Acad. Sci. U.S,A. 66, 1157 (1970). * An apparent exception to this is the P-450 (P-450~c) isolated from adrenal mitochondria and involved in the conversion of cholesterol to pregnenolone. This cytochrome is predominantly high spin as isolated; however, the isolated preparations contain at least a stoichiometric amount of cholesterol so that this high-spin species may be the P-450~ccholesterol complex. 5 A. Y. H. Yu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 44, 3714 (1969). e j. A. Canick, K. J. Ryan, N. R. Orme-Johnson, W. H. Orme-Johnson, and A. C. Brownie, Meeting of the Endocrine Society, 58th, 1976, San Francisco, Calif., p. 113. 7 C. R. Jefcoate, R. Hume, and G. S. Boyd, FEBS Lett. 9, 41 (1970). s Even when an excess of a ligand is present, there may be an equilibrium between the high-spin and low-spin forms so that measuring the amount of P-450 in the predominant spin form will not yield the total amount. However, in the cases for which this phenomenon has been quantitated, the amount in the alternate spin state is less than 10% of the total amount. Therefore, quantitation of the predominant form yields numbers that are very close indeed to the true value, and, quantitation of both spin forms gives the true value [S. G. Sligar, Biochemistry 15, 5399 (1976)].
[26]
253
DETECTION AND QUANTITATION OF CYTOCHROME P-450
79* K 245
15 ° K tge
I
2~15226 1~t911/~
450
PHE-
.IM ) y_)/ f~S----
P450 ÷ CAMPHOR W
FIG. 1. Electron paramagnetic spectra of Pseudomonas putida P-450 at 79 ° and 15°K. Top: 1.1 rnM P-450 untreated; center: after addition of 0.25 volume of a 6 mM solution of 1phenylimidazole; bottom: after addition of 0.25 volume of a 6 mM solution of D-(+)camphor. The conditions of EPR spectroscopy were at 79 ° and 15°K, respectively: microwave power, 27 and 0.27 mW; scanning rate 160 and 625 G/min; modulation amplitude 6 G and time constant 0.25 sec throughout. The amplification of the center and bottom curves, recorded at 79°K, is 1.25 times that of the top curve to correct for dilution and the amplification of the bottom curve recorded at 15°K is 3.1 times that of the others. From R. Tsai, C. A. Yu, I. C. Gunsalus, J. Peisach, W. Blumberg, W. H. Orme-Johnson, and H. Beinert, Pro('. Natl. Acad. Sci. U.S.A. 66, 1157 (1970).
few specific examples may hint at the enormous amount of information available from EPR spectra. P-450scc presents a more complicated picture when bound to substrate, postulated reaction intermediates, and product. P-450~c bound to cholesterol is high spin; bound to 20-ahydroxycholesterol or 22-R-hydroxycholesterol is low spin; bound to 20,22-dihydroxycholesterol is high spin; and bound to pregnenolone is low spin. Further it is possible to determine from the EPR spectra which of the steroids producing a low-spin complex is bound. 9 Similarly, high9 N. R. Orme-Johnson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1246, Abstr. 124 (1974).
254
MICROSOMAL ELECTRON TRANSPORTAND CYT P-450
[26]
spin P-450 complexes do not all have the same g values. For example in rat adrenal mitochondria, the P-450~l,.deoxycorticosterone complex has gmnx = 7.9 while the P-450~e'cholesterol complex has gmax = 8.1, so that it is possible to monitor these two species separately in the intact organelles,l° though the spectra are overlapped and may require spectral simulation for satisfactory quantitation.
Low-Spin Heine---Detection and Quantitation EPR spectra of low-spin cytochrome P-450 are easily observed at temperatures
