Substrate-induced Difference Spectra Research Articles

Interaction between Mitochondrial CYP27B1 and Adrenodoxin: Role of Arginine 458 of Mouse CYP27B1

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.

Structure–function analysis of CYP27B1 and CYP27A1

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.

Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T).

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

Structure-related inhibitory effect of antimicrobial enoxacin and derivatives on theophylline metabolism by rat liver microsomes.

Enoxacin, an antimicrobial fluoroquinolone with a 7-piperazinyl-1, 8-naphthyridine skeleton, is a potent inhibitor of cytochrome P-450-mediated theophylline metabolism. The present study was designed to clarify, using seven enoxacin derivatives, the molecular characteristics of the fluoroquinolone responsible for the inhibition. Three derivatives with methyl-substituted 7-piperazine rings inhibited rat liver microsomal theophylline metabolism to 1,3-dimethyluric acid to an extent similar to that of enoxacin (50% inhibitory concentrations [IC50s] = 0.39 to 0.48 mM). 7-Piperazinyl-quinoline derivatives, 8-hydroenoxacin (8-Hy) and 1-cyclopropyl-8-fluoroenoxacin (8-F1), which have a hydrogen and a fluorine at position 8, respectively, more weakly inhibited metabolite formation (IC50s = 0.88 and 1.29 mM, respectively). Little inhibition (IC50 > 2 mM) was observed in those with 3'-carbonyl and 4'-N-acetyl groups on the piperazine rings. The substrate-induced difference spectra demonstrated that the affinities of enoxacin, 8-Hy, and 8-F1 to cytochrome P-450 were parallel with their inhibitory activities. The substituent at position 8 was found to determine the molecular conformations of the fluoroquinolones, and the planarity in molecular shape decreased in the same order as the inhibitory activity (enoxacin > 8-Hy > 8-F1). Moreover, the 3'-carbonyl and 4'-N-acetyl groups decreased the basicity of their vicinal 4'-nitrogen atoms when judged from their electrostatic potentials, which showed a remarkably broadened negative charge around the nitrogens. As a result, the planarity of the whole molecule and the basicity of the 4'-nitrogen atom of enoxacin are likely to be dominant factors in the inhibition of theophylline metabolism by cytochrome P-450.

Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.

We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17 alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11 beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373-->Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17 alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373-->Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.

Purification from Adult Pig Testicular P-450 and 17α-Hydroxylase Activity of P-450 Containing Liposomal Membranes

A cytochrome P-450 from adult pig testicular microsomes was purified to a specific content of 12 nmol P-450/mg protein. P-450 has a minimum molecular weight of 46000 ± 1000, as judged on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Adult testicular P-450 is prepared in the low-spin form with an absorbance maximum at 417 nm. The substrate-induced difference spectrum with progesterone is a typical I difference spectrum. P-450 was incorporated into liposomal membranes composed of phosphatidylcholine, and 17 α-hydroxylase activity was shown to amount to 15.5 nmol product/min/nmol of P-450. Furthermore, testicular cytochrome b 5 did not increase the 17 α-hydroxy1ase activity, and the activity was largely inhibited by the addition of sodium cholate, Emulgen 913 and testicular lipid.

Spectrophotometric and electron spin resonance studies on the substrate interactions of ferredoxin-linked nitrite reductase from spinach.

Interactions of ferredoxin-linked nitrite reductase (NiR) from spinach with its substrate were studied by spectrophotometry and electron spin resonance (ESR) spectroscopy. Siroheme was extractable from NiR with 2.5% (W/V) trichloroacetic acid (TCA) and with acetone containing 0.01 N HCl. The addition of nitrite or sulfite to these extracts resulted in shifts of the absorption spectra of siroheme. The HCl-acetone extract showed ESR signals of symmetrical high spin heme, which disappeared on addition of nitrite. Spectral titration indicated a high affinity of extracted siroheme to nitrite and sulfite. The addition of nitrite or sulfite to protoheme dissolved in 0.01 N HCl-acetone did not cause a shift of the absorption spectrum. The extractability of siroheme with 0.01 N HCl-acetone was suppressed by the addition of nitrite to the NiR preparation. Moreover, a substrate-induced difference spectrum with peaks at about 295 and 287 nm was observed on addition of nitrite to NiR. These observations indicated an intrinsic strong affinity of siroheme to nitrite and sulfite, formation of rhombicity of siroheme by binding to the protein moiety, and also a probable conformational change of NiR on binding to the substrate. In agreement with previous reports, ESR signals of the heme-NO complex were observed with NiR in the presence of nitrite, methyl viologen (MV), and dithionite. In the present study, the same signals of similar intensity were also observed on omission of MV, under which conditions no catalytic reduction of nitrite occurred. Furthermore, the signal of the heme-NO complex was not observed when MV was replaced by spinach ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Zonal distribution of cytochrome P-450s (P-450 11β and P-450 scc) and their relation to steroidogenesis in bovine adrenal cortex

Zona glomerulosa and zonae fasciculata-reticularis of bovine adrenal cortex were separated by our improved method using a remodeled microtome. A considerable amount of fresh mitochondria and microsomes was quickly obtained. Specific content of cytochrome P-450 in the mitochondria was higher in zonae fasciculata-reticularis than in zona glomerulosa. When metyrapone was added to the mitochondria and the drug-induced difference spectra were measured, there appeared two phases in the titration curve; the first phase was saturated at a concentration of 3 μM and the second phase appeared at concentrations higher than 10 μM. The first phase seemed to be due to the substrate-free P-450 11β and the second phase to the cholesterol-bound P-450 scc. From these spectral studies contents of cytochrome P-450 scc and P-450 11β were estimated, and those were found to be equal for each zone. From similar studies on substrate-induced difference spectra, there seemed to be no difference between two zones in the affinity of cytochrome P-450 11β for deoxycorticosterone and 11-deoxycortisol. This was also the case with 11β-hydroxylase activity and substrate specificity for deoxycorticosterone and 11-deoxycortisol.

The recognition of a soluble cytochrome P450 in rat liver

The presence of hepatic soluble cytochrome P450 was investigated in the rats that were pretreated with CCl 4, CS 2 and DENA + PCBs. The dithionite-reduced CO difference spectrum of the hepatic 204000 × g supernatant of these rats showed its maximum at 455 nm. Aniline and hexobarbital could react with the hepatic 204000 × g supernatant to give the substrate binding spectra of P450; differences were observed between the substrate-induced difference spectra of the 204000 × g supernatant and that of the microsomal fraction. These results indicate the existence of a hepatic soluble P450.

Characteristics of microsomal enzyme controls in primary cultures of rat hepatocytes

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[ a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ max = 447.9 nm). However, metabolism of benzo[ a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.

Effect of lanthanons on substrate-induced difference spectra in rat liver microsomes

Intravenously administered light lanthanons change spectral interactions in rat liver not only by decreasing the concentration of cytochrome P-450, but they also cause a qualitative change in the cytochrome P-450 molecule or its microenvironment.

Substrate-induced Difference Spectral, Electron Paramagnetic Resonance, and Enzymatic Properties of Cholesterol-depleted Mitochondrial Cytochrome P-450 of Bovine Adrenal Cortex

Abstract The steroid substrate-induced difference spectra, electron paramagnetic resonance (EPR) properties, reduction of cytochrome P-450, and hydroxylation activities were studied in acetone-extracted mitochondrial preparations of bovine adrenal cortex. Cholesterol, 20α-hydroxycholesterol, 22R-hydroxycholesterol, 17α-hydroxypregnenolone, progesterone, deoxycortisol, and deoxycorticosterone all exhibited a type I difference spectrum with characteristic K's values. These preparations were active in side chain cleavage of cholesterol and 20α-hydroxycholesterol, as well as 11β-hydroxylation of deoxycortisol. Additive type I spectral changes were observed with cholesterol and deoxycortisol while competitive inhibition and noncompetitive inhibition were found with cholesterol in the presence of 20α-hydroxycholesterol and pregnenolone, respectively. EPR studies showed a low to high spin transition of Fe3+ in P-450 with cholesterol or 20α-hydroxycholesterol. Preincubation of cholesterol with the acetone powder preparations caused 20α-hydroxycholesterol, 22R-hydroxycholesterol, and pregnenolone to induce the modified type II difference spectra and high to low spin transition in Fe3+ of P-450. A correlation between decreases in low spin signal area and increases in the type I spectral changes was found in cholesterol titration studies. Triton N-101 markedly inhibited the formation of type I difference spectra, the formation of high spin P-450, and the activity of side chain cleavage of cholesterol. The quantitative correlation between these inhibitory effects of Triton N-101 on P-450 suggests that the formation of high spin P-450 by its substrate is an obligatory event in the side chain cleavage of cholesterol. A slow control rate of P-450 reduction by NADPH in the presence of flavoprotein reductase and adrenodoxin was markedly stimulated by all of the substrates tested. The results suggest that substrate-induced high spin formation of P-450 appears to be an essential step for acceptance of reducing equivalents for oxygen activation and hydroxylations of steroid substrates.

Further studies on the P-450 difference spectra induced by various compounds in liver microsomes from 3-methylcholanthrene treated rats.

Substrate induced difference spectra in microsomes from 3-methylcholanthrene treated rats were investigated using substrates which cause type I difference spectrum in microsomes from control rats. It was found that α, α'-dipyridyl showed only type II spectrum, whereas SKF 525-A and ethylmorphine produced type I spectra. Additionally, hexobarbital, aminopyrine and imipramine produced type I spectra when they were used by rather low concentrations but type II spectra were induced by high concentrations of them.α, α'-Dipyridyl caused type II spectrum when microsomes from control rats were treated with phospholipase C. The type II spectrum of α, α'-dipyridyl in 3-methylcholanthrene-induced microsomes was increased by treatment with phospholipase C.Moreover, α, α'-dipyridyl showed type II spectrum in microsomes from control rats when SKF 525-A, which has a high affinity to combine with type I binding site of P-450, was added into reference and sample cuvettes.From above data, the authors concluded that most of type I compounds could combine with both type I and type II binding sites, and discussed on the properties of type I and type II binding sites.

Substrate-induced difference spectra and cholesterol to pregnenolone conversion with adrenal heme protein P-450.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSubstrate-induced difference spectra and cholesterol to pregnenolone conversion with adrenal heme protein P-450Shlomo Burstein, Nana Co, Marcel Gut, Heinz Schleyer, David Y. Cooper, and Otto RosenthalCite this: Biochemistry 1972, 11, 4, 573–577Publication Date (Print):February 15, 1972Publication History Published online1 May 2002Published inissue 15 February 1972https://doi.org/10.1021/bi00754a015RIGHTS & PERMISSIONSArticle Views41Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (604 KB) Get e-Alerts Get e-Alerts

Soluble cytochrome P.450 from bovine adrenocortical mitochondria

A method is given for solubilizing at least one species of cytochrome P.450 from bovine adrenocortical mitochondria. The soluble P.450 supports side-chain cleavage of cholesterol but not 11β-hydroxylation, thus clearly separating these two activities for the first time. Soluble P.450 shows a substrate-induced difference spectrum with both cholesterol and 11-deoxycorticosterone; these difference spectra are opposite in type (peak at 420 nm) to spectra observed when these substrates are added to crude P.450. It is suggested that there are at least two cytochromes P.450 in bovine adrenocortical mitochondria with distinct enzymatic activities namely, side-chain cleavage and 11β-hydroxylation.

Diphenylhydantoin-induced difference spectra with rat-liver microsomes

Addition of diphenylhydantoin (DPH) to rat-liver microsomes caused a type l substrate-induced difference spectrum with a peak at 390 nm and a trough at 425 nm. The magnitude of this substrate-induced change, δA max was relatively small (mean absorbance: 0.027 ± 0.003 S.E., N = 6 per 3 mg of microsomal protein per ml) as compared with that caused by benzphetamine or aniline. The apparent K s for the DPH-inicrosome interaction was low: 27 ± 2 μM(mean ± S.E., N = 6). The δA max of substrate-induced difference spectra caused by addition of DPH to microsomes was proportional to the microsomal protein concentration, but rapidly decayed during storage at 4° (80% reduction in 24 h). Treatment of animals with phenobarbital, DPH, chlordane, or DDT caused a reduction of δA max of the DPH-microsome interaction as compared with that obtained with hepatic microsomes from the control animals. Treatment of animals with 3-methylcholanthrene (3MC) either abolished the δA max of DPH-microsome interaction or caused a reversal to a modified type II response. Addition in vitro of phenobarbital, DPH, chlordane, DDT, or 3MC as modifiers to the microsomes from control animals, reduced or abolished the δA max of substrate-induced difference spectra with DPH as substrate. It may be concluded that DPH despite its low K s is a rather weakly binding substrate which is easily replaced by more strongly binding ligands.

Effects of storage on hepatic microsomal cytochromes and substrate-induced difference spectra

Microsomal cytochrome P450, the ethyl isocyanide difference spectrum of dithionite-reduced microsomes (measured at the pH giving equal absorbance of 430 and 455 mμ peaks), and the aniline type II difference spectrum were affected similarly after phenobarbital induction and during the subsequent aging in vitro (about 90 hr at 1° under nitrogen) of hepatic microsomes from rats pretreated with phenobarbital. In these same microsomal preparations from phenobarbital-pretreated rats, type I spectra (caused by adding hexobarbital or (+) benzphetamine to microsomes) were increased to a greater extent and were less stable to aging in vitro than the previously mentioned components. During aging, decreases in the type II spectra paralleled the loss of total heme, but large decreases in the type I spectra were observed with little or no loss of heme. Also, aging did not change the pH giving equal absorbance of the 430 and 455 mμ peaks in the ethyl isocyanide difference spectrum, nor was there any shift in the wavelength of the maximum (450 mμ) in the carbon monoxide difference spectrum of dithionite-reduced microsomes. The binding associated with type I or II spectral changes did not appear to conform to simple Michaelis-Menten kinetics. Plots of ΔA vs. ΔA/S (where ΔA is the difference in absorbance between the trough and the peak in the 350 to 450 mμ region of the type I or II substrate-induced microsomal difference spectrum, and S is the corresponding substrate concentration) exhibited curvature (concave upwards). During aging, the curvature associated with plots of ΔA vs. ΔA/S using ( + ) benzphetamine increased, while the curvature associated with plots of ΔA vs. ΔA/S using aniline decreased. Some correlation was observed between increases in the rate of N-demethylation of (+) benzphetamine or p-hydroxylation of aniline and the increases in the respective type I or II difference spectra, after phenobarbital treatment of rats. Also it appeared that the rate of p-hydroxylation of aniline decreased to a lesser extent on aging than the rate of N-demethylation of (+) benzphetamine.

Substrate induced spin state changes in cytochrome P450

Deoxycorticosterone (DOC) produces 385 and 645 nm peaks and 420, 530 and 575 nm troughs, and 20αOH-cholesterol produces 420, 530 and 575 nm peaks and 385 and 645 nm troughs in the difference spectra of adrenal cortical submitochondrial particles. Electron spin resonance studies show that 20αOH-cholesterol produces an increase and 11-deoxycortisol or DOC produce a decrease in the low-spin signal of P450. These results suggest to us that the substrate induced difference spectra may be the result of a spin state shift in cytochrome P450.

Studies on P-450: V. On the Substrate-Induced Spectral Change of P-450 Solubilized from Bovine Adrenocortical Mitochondria<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>

Procedures for solubilization and purification of P-450 from bovine adrenocortical mitochondria were examined and an improved method for the preparation of partially purified P-450 was described. By measuring the “substrate-induced difference spectrum”, the substrate-binding specificity of the solubilized preparation was compared with those of P-450 in bovine adrenocortical mitochondria, bovine adrenocortical microsomes, and rat liver microsomes. The specificity of the solubilized preparation was mostly the same as that of P-450 in adrenocortical mitochondria. The specificity of mitochondrial P-450 and that of P-450 in adrenocortical microsomes were similar in qualitative aspects, but the relative intensity of spectral change and the affinity for substrate were frequently different. On the other hand, the binding specificity of hepatic microsomal P-450 was rather different qualitatively from those of adrenal P-450's. The effect of successive addition of two different substrates to the solubilized preparation was also examined. The intensity of spectral change induced by a type I substrate was inhibited non-competitivcly by the addition of another type I substrate, but was not affected by the addition of a type II substrate. The significance of these results was discussed. In addition to these observations, spectral evidence was presented indicating the change in spin state of P-450 by binding with a substrate.