of biological chemistry vol. no. of in heme ligand ... · 3074 ligand replacement reactions of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 257, No. 6, Issue of March 25, pp. 3073-3083, 1982

Heme Ligand Replacement Reactions of Cytochrome P-450 CHARACTERIZATION OF THE BONDING ATOM OF THE AXIAL LIGAND TRANS TO THIOLATE AS OXYGEN*

(Received for publication, September 8,1981)

Ronald E. White$ and Minor J. Coon From the Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, Michigan 48109

Evidence of several types has accumulated that cytochrome P-450 has a thiolate anion as one of the axial ligands to heme (the fifth ligand). On the other hand, there is as yet no general agreement on the nature of the axial ligand trans to thiolate (the sixth ligand), although nitrogen and oxygen have been proposed. To resolve the controversy, the ligand exchange reactions of cytochrome P-450 were investigated by the use of optical spectroscopy. Two isozymes of rabbit liver microsomal cytochrome

P-450 were examined: the isozyme induced by phenobarbital (P-450mz), which has a sixth ligand and is low spin and the isozyme induced by 5,6-benzoflavone (P- 45Om4), which is without a sixth ligand and is high spin. A series of artificial ligands was chosen to model the coordination of each of the potential native ligands, including water and certain amino acid residues. When the artificial ligand coordinated through an oxygen atom, the spectrum of pentacoordinate P - 4 5 0 ~ ~ changed to one closely resembling that of native, hexacoordinate P-450~2 . The spectrum of P - 4 5 0 ~ ~ was unchanged in the presence of oxygen-coordinating ligands. However, when artificial ligands which coordinated through nitrogen or sulfur were added to either P - 4 5 0 ~ ~ or P-450m4, the induced spectra did not resemble the native spectrum and, in fact, were distinctive and characteristic of the particular ligand type. With three of the artificial ligands, 1-butanol, l-benzylimidazole, and diethylphenylphosphine, the binding was found to be reversible by dilution, ultrafiltration, or gel filtration. The binding of 1-pentanol and l-benzylimidazole was competitive, as expected for heme ligands, and the number of 1-benzylimidazole binding sites per molecule of P-450mz was estimated as 1.1. These results provide strong evidence that the native sixth ligand in P - 4 5 0 ~ ~ is oxygen rather than nitrogen.

Since cytochrome P-450 was first identified as a hemeprotein (2), continuing attempts he been made to establish the nature of the nonporphyrin ligands to the heme iron atom. One of these ligands, termed the fifth ligand, is trans to carbon monoxide in the ferrous carbonyl state of cytochrome P-450 and is probably present throughout the catalytic cycle of the

* This research was supported by Grant PCM76-14947 from the National Science Foundation and Grant AM-10339 from the United States Public Health Service. A preliminary account of part of this investigation has been presented (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

# Present address, Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032.

enzyme. The unusually long wavelength of the Soret absorption (approximately 450 nm) in the spectrum of ferrous carbonyl P-450 has prompted the idea that the fifth ligand is unlike those found in other, non-P-450 hemeproteins. Fur- thermore, the native, ferric protein, when in the low spin state, exhibits uniquely low anisotropy in the EPR spectrum, but in the high spin state shows the greatest rhombicity of any high spin hemeprotein known. The presence of a ligated sulfur anion, first suggested by Mason et al. (3), was invoked to rationalize the atypical aspects of both the optical and EPR spectra of the protein. Shortly thereafter, Roder and Bayer (4), using hemin-thiol complexes, and three other groups, Jefcoate and Gaylor (5), Hill et al. (6), and Blumberg and Peisach (7), using thiol complexes of hemoglobin and myoglobin, confirmed that low spin hemin-thiolate complexes did indeed exhibit EPR spectra similar to that of cytochrome P- 450. Consequently, many investigators have searched for such a ligand in the native protein (8-14) or have developed thiolate-based models which reproduce various spectral features of the protein (15-22). The synthetic models have been particularly persuasive in favor of thiolate ligation. The optical spectrum of ferrous carbonyl cytochrome P-450 was fist recognized as a hyperporphyrin spectrum by Hanson et al. in 1976 (23). Thus, as they pointed out, the long wavelength Soret band of the ferrous carbonyl derivative is merely the low energy limb of a split Soret peak, the high energy limb appearing at about 363 nm. Furthermore, these authors presented molecular orbital calculations which predicted such splitting when the ligand trans to CO was thiolate but not thiol. However, the most convincing evidence so far for the presence of a thiol axial ligand in cytochrome P-450 has come from the powerful new technique extended x-ray absorption fine structure. By this method, not only is the presence of a thiolate ligand established, but an Fe-S bond length of 2.19 8, may even be assigned in the native protein (24). A more complete analysis of the evidence for thiolate ligation is presented elsewhere (25, 26).

The nature of the ligand trans to thiolate (the sixth ligand), although perhaps no less important to the enzyme function- ally, has received less attention. Two general types of ligands have been proposed, depending on whether coordination to iron occurs through a nitrogen or an oxygen atom. A nitrogen ligand is most frequently pictured as the side chain imidazole of a histidyl residue of the protein, and a number of research- ers have taken this view (5, 10, 19, 27, 28). Most recently, Peisach et al. (29) reported the electron spin echo envelope of cytochrome P-450, which shows nuclear modulation of the EPR signal of low spin ferric iron. These authors felt that the modulation most likely arose from the remote nitrogen of an iron-coordinated imidazole. That the native sixth ligand might be oxygen was first proposed by Griffin and Peterson (30) and later by several others (20, 31-34).

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3074 Ligand Replacement Reactions of Cytochrome P-450

We wish to report the results of a study of the ligand exchange reactions of two purified forms of rabbit liver microsomal cytochrome P-450 (P-4501,~).' In particular, we present evidence that the sixth ligand to iron in low spin ferric P- 4501,M2 is of the oxygen type. However, we are unable to distinguish a coordinated water molecule from a protein-donated oxygen ligand by these experiments. On the other hand, the evidence does indicate that the native sixth ligand cannot be nitrogenous, such as imidazole or amine, nor can a thiol or thioether be considered in the sixth position.

MATERIALS AND METHODS

1-Benzylimidazole (Aldrich Chemical Co.) was sublimed to yield white crystals, m.p. 73-73.5 "C. 2-Phenylimidazole was a gift of Dr. John H. Dawson, University of South Carolina, and was twice recrys- tallized from benzene to yield white crystals, m.p. 146.5-147 "C. 4- Picoline and 2,4-lutidine (Aldrich) were distilled immediately before use. Diethylphenylphosphine was obtained from the Alfred Bader Chemicals division of Aldrich Chemical Co. Phenylcyclohexane was prepared according to the procedure of Corson and Ipatieff (35). Benzyl isocyanide (Aldrich) was used rather than the more commonly employed ethyl isocyanide because the former compound has a greater affinity for P-4501,~ and has a considerably less disagreeable odor. Nitrogen was prepurified grade while carbon monoxide was c.p. grade. BASF catalyst was obtained from ACE Glass Co. 5-Deazari- boflavin was a gift from Dr. Vincent Massey, The University of Michigan. Nitric oxide was prepared as described previously (36).

Phenobarbital-inducible P - 4 5 0 ~ ~ ~ and 5,6-benzoflavone-inducible P-4501.~~ were prepared from rabbit liver microsomes by the procedure of Coon et al. (37). The specific contents were typically about 16 nmol/mg of protein for P-4501.~2 and 15-18 for P-450LMr. The denatured P-420 derivatives of these proteins were prepared either by exposure to 1 mM sodium dodecyl sulfate or to 1.1 M potassium thiocyanate. The conversion by the detergent was almost instanta- neous, while the chaotropic salt required at least 30 min at 25 "C. When sodium dodecyl sulfate was to be used, the buffer was sodium rather than potassium phosphate. Concentrations (in micromolar) of P-450 and P-420 in mixtures were estimated by the use of Equations 1-4 derived from the micromolar absorptivities of the P-420 and P- 450 forms at 423 and approximately 450 nm. Because the exact molar absorptivity of P-420-free P-4501.M at 423 nm is uncertain,

[P-4501.M2] = 9.3A451 - 0.57Ad~ (1)

[P-420l,M2] = 5.2A42, - 2 A 4 5 1 (2)

[P-450~~41 = 8.6A4.17 - 0.59Ac2:3 (3)

[P-42ol,M4] = 514423 - 1.8A.147 (4)

these equations probably are less reliable for determining the concentration of P-420 when the ratio P-420/P-450 is less than 0.1. Absor- bance spectra were determined with either a Varian-Cary 219 or an Aminco DW-2 recording spectrophotometer. The monochromators were calibrated by reference to the 486.0- and 656.1-nm emission lines of the deuterium lamp. A spectral band width of 1.0 nm was used. Quartz cuvettes of 1-cm path length were employed. Molar absorptivities (absorption coefficients) are given in units of M" cm" in the figures and m"' cm" in the tables.

Reactions of cytochrome P-450 were carried out at 25 "C in 0.1 M potassium phosphate buffer, pH 7.4 (standard buffer). Unless other- wise noted, P - 4 5 0 ~ ~ ~ and P-4501.~4 solutions always contained 80 pM and no dilauroylglyceryl3-phosphorylcholine, respectively. The phospholipid was added as a 1 mg/ml sonicated suspension in water. The presence or absence of phospholipid did not affect the nature of the spectral change brought about by a particular substance, but usually did increase the apparent affinity of the substance for P-45oLM.

The abbreviations used are: P-4501.~, liver microsomal cytochrome P-450; P-4201.~, denatured derivative of P"i501,M with altered spectral properties and no biological activity; P-450,.,,, soluble cam- phor-hydroxylating cytochrome P-450 isolated from Pseudomonas putida. The isozymes of rabbit liver microsomal cytochrome P-450 are numbered according to their electrophoretic mobilities; P-450LMZ is the isozyme induced by phenobarbital, while P-450I.M4 is the isozyme induced by 5,6-benzoflavone.

Substances to be tested as ligands to P - 4 5 0 ~ ~ were added as concentrated solutions in methanol or water or, when possible, as the pure materials.

Samples were deoxygenated in anaerobic cuvettes by alternate evacuation and flushing with oxygen-free nitrogen or carbon monoxide. The liquid phase was equilibrated with the anaerobic gas after each flush by tipping the cuvette and gently agitating the contents. Six to ten such cycles were performed. Nitrogen and carbon monoxide were freed of oxygen by passage through a column of reduced BASF catalyst maintained at 120 "C.

Reductions with dithionite were accomplished by injecting about 10 pl of 0.2 M sodium dithionite in 0.1 M potassium pyrophosphate buffer, pH 8.5, into the previously degassed sample in a capped cuvette. Occasionally, a few crystals of solid sodium dithionite were added instead from a side arm of the anaerobic cuvette, but this method gave slower reduction and was more destructive to the P-450. For photoreductions (38), the protein solution containing added EDTA (0.3 mM) was deoxygenated, a quantity of 5-deazariboflavin sufficient to give about 1 p~ final concentration was added from a light-protected side arm of the anaerobic cuvette, and irradiation was conducted in an ice bath with a 350-watt tungsten halogen lamp at a distance of 5 cm. The sample was irradiated in 1- to 2-min intervals and the extent of reduction was monitored after each. Usually 5-10 min of total irradiation was sufficient for the reduction. The 5-deazariboflavin may be replaced with an equal concentration of pro- flavin (3,6-diaminoacridine). Photoreduction has the advantage over dithionite reduction that absorbances in the region below 400 nm are not obscured.

Apparent dissociation constants were estimated by analysis of absorbance changes as a function of ligand concentration using double reciprocal plots. The expressed dissociation constants are the apparent values and have not been corrected for the pH effect on the true concentrations of ligating species such as cyanide or alkylamine. Correction for substrate depletion effects was accomplished by an iterative procedure.

RESULTS

For comparison with other spectra to be presented, the absorption spectra of P - 4 5 0 1 , ~ ~ and P - 4 5 0 1 . ~ ~ are shown in Figs. 1 and 2 and the maxima and millimolar absorptivities are included in Tables I and 11. While these two isozymes show essentially the same spectra in the ferrous and ferrous carbonyl states, the spectra of the ferric states are profoundly different. The spectrum of P-450LM2 has the Soret band at 417 nm and exhibits distinct a- and @-bands in the visible region. On the other hand, the spectrum of P-450LM4 shows a blue- shifted Soret band at 393 nm, no distinct a- and ,&bands, and a weak band at 645 nm which may be assigned to a charge- transfer transition and is characteristic of high spin hemepro-

I I I I 1 I 1201

1 I I I I I I 350 400 450 500 550 600 650

WAVE LENGTH Inml

FIG. 1. Absorption spectra of P - 4 5 0 ~ ~ ~ in various states. -, Native, ferric P-4501.M2 (3.9 p ~ ) ; - - -, ferrous P-4501,MZ prepared by anaerobic photoreduction as described under "Materials and Methods." . . . . , Ferrous carbonyl P-4501.~~. The anaerobic cuvette containing the ferrous cytochrome was refilled with carbon monoxide while maintaining anaerobiosis. . -. , Ferric, phenylcyclohexane- bound P-4501,w2. To the native cytochrome (3.9 p ~ ) was added phenylcyclohexane, final concentration 50 p ~ , as a 1 M solution in methanol.


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Ligand Replacement Reactions of Cytochrome P-450 3075

I I I 2, teins (40). In fact, the spectral differences in these two proteins I I I 120 -

arise from the fact that P - 4 5 0 ~ ~ 2 is low spin in the ferric state, ~ 2 0 while P-4501,~~ is high spin, a conclusion confirmed by the

EPR spectra (39). It is clear from studies of myoglobin (36) ~ 16 and of models (19) that the iron in low spin hemeproteins is

hexacoordinate and approximately octahedral, while the iron ,2 in high spin hemeproteins is pentacoordinate and square

pyramidal. Thus, the essential difference between P-4501.M2 - and P-45oLM4 is the absence of a sixth or distal ligand trans to

thiolate in the latter protein. The sixth ligation position of P-

Lewis bases, albeit with a lower affinity than is displayed with P-450LM2. Thus, it is possible to investigate the coordination

F ~ ~ . 2. Absorption spectra of P&iOLM4 in various states. compounds to pentacoordinate P - 4 5 0 ~ ~ 4 in order to mimic the -, Native, ferric p-45OLM4 (3.9 p ~ ) ; - - -, ferrous p-450LMr; . . . . , various potential candidates (ie. amino acid side chains and ferrous carbonyl P -450~~4 . water) for the native sixth ligand in ferric P"t50LM2.

20 - , 4 5 0 ~ ~ ~ is, nonetheless, available for coordination of suitable

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WAVELENGTH lnml sphere of hexacoordinate P-4501,~~ by the addition of certain

TABLE I Absorbance spectra of ferric cytochrome complexes

The abbreviations used in the table are: BHA, 0-benzylhydroxylamine; BIC, benzyl isocyanide; BzlIm, 1-benzylimidazole; Im, imidazole; MeIm, 1-methylimidazole; PMS, pentamethylene sulfide; 2-PhIm, 2-phenylimidazole; OctNHs, octyl amine; OctSH, octane thiol; and s, shoulder.

Added ligand

None None None None

1-BuOH

Im Im MeIm MeIm BzlIm BzlIm BzlIm 2-PhIm 2-PhIm OctNHz OctNHz BHA BHA Picoline NO NO NB-

OctSH PMS PMS NCS-

BIC BIC BIC CN- CN

EtsPhP Et2PhP

P - 4 2 0 ~ ~ 2 EtnPhP " Values in parentheses are the millimolar absorptivities (m"' cm").

n.u.v.h nm

361 (43)

366s 366s

362

359 (45) 356 360 (41) 357 359 (45) 361 (55) 360 (39) 357 (41) 362 (46) 361 (47) 367 (49) 360 (42)

360 (50) 359 (63) 357 (76) 366 (56)

379 361 (59) 365 (56) 361 (42)

362 (48) 364 (49) 359 (33) 365 (49) 368 (60)

377 377 377

-

*

Absorbance maxima"

Soret P nm nrn

418 (110) 535 (15) 393 (104) 416 (97) 540 (11) 418 (81) 540 (12)

417 533

426 (104) 424 424 (102) 541 (12) 424 539 423 (104) 540 (15) 423 (101) 539 (16) 415 (105) 419 (109) 536 (13) 418 (98) 535 (17) 423 (102) 540 (16) 424 (100) 423 (104) 423 (85) 422 (104) 536 (17) 433 (98) 543 (19) 433 (101) 542 (12) 421 (86) 538 (15)

469 424 (94) 541 (19) 425 (85) 538 (16) 419 (94) 539 (14)

430 (99) 430 (98) 432 (179) 533 (14) 436 (73) 436 (66)

452 450 452

-

*d

*

*

* * *

-

- -

- -

- - -

a nm

569 (16) 645 (8.8) 564s 570s

567

* *

575s 578s 574s 575s

570 (13) 569 (15) 572 (13)

*

* * *

575s 575 (16) 571 (9) 570s

554 570s 570s 570s

547 (16) 547 (16) 563 (12) 559 (14) 553 (16)

560 -550

552

Ligand concentration

Apparent Kc, of ligand

mM

250

50 200

32 160

0.050 1.3 0.2

15 17 0.5

0.5 4.3

10'

39 -1 -1

1600

1 1.6

10 920

0.25 2 0.8

50 90

0.5 0.33 0.5

mm

100

5 55'

6 40 0.001 0.05'

6 0.02

0.16

3 1.2'

200

4 350

0.02 0.2 0.3 7

20'

' Near ultraviolet. Dash indicates absence of band in that region. Asterisk indicates presence or absence of band not determined. 160 p~ phospholipid.

'Ref. 39.


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TABLE I1 Absorbance spectra of ferrous cytochrome complexes

Absorbance maxima Ligand Cytochrome con-

centration n.u.v." Soret a

Native P - 4 5 0 ~ ~ 2 None P - 4 5 0 ~ ~ ~ None P-4201,~e None P -4201 .~~ None

Nitrogen P-450LM2 BzlIm" P - 4 5 0 ~ ~ 4 BzlIm P-4201.~2 BzlIm P"fi01.M2 ImE

Carbon P - 4 5 0 ~ ~ 2 CO P-4501.~4 CO P - 4 2 0 ~ ~ 2 CO P - 4 2 0 ~ ~ 4 CO P - 4 5 0 ~ ~ 2 BICh P-4501,~r BIC'

Phosphorus P-450~~1 EtlPhP P - 4 5 0 ~ ~ 4 EtePhP P-4201,wr EtrPhP

nm nm

- 413 (88)' - 544 (20) - 411 (90) - 542 (20) 325 (40) 428 (109) 535 (12) 562 (19) 320 (63) 427 (106) 531 (11) 558 (16)

358s 421 (86) - 547 (17) *' 422 (92) - 538 (19) * * 426 (136) * $

427 (98) * *

368 (60) 451 (110) - 554 (19) 366 (63) 447 (118) - 549 (19) 341 (35) 423 (195) 541 (15) 570 (15) 321 (55) 423 (207) 541 (15) 570 (15) *

* 432/456 - 552 433/451 *

457 552 580s 455 552 580s 457 552 578s

*

* t

*

mM

0.05 1.3/ 0.2

50

-1 -1 -1 -1

0.25 2

0.5 0.33 0.5

" Near ultraviolet. ' Dash indicates absence of band in that region. 'Values in parentheses are the millimolar absorptivities (m"'

'' I-Benzylimidazole. ' Asterisk indicates presence or absence of band not determined. ' 160 PM phospholipid.

' Benzyl isocyanide.

One may partially convert the spectrum of low spin ferric P-4501.~2 to one resembling high spin P-4501,~~ by the addition of a variety of hydrophobic compounds which cannot coordinate at the iron. This phenomenon is the basis for the well- known type I substrate-binding difference spectrum (41). An example of such a spectrum is the one induced with P-45oLM2 by phenylcyclohexane (Fig. 1). An absorbance shoulder has formed at 390 nm to the detriment of the 418 nm band; the distinct a- and P-bands have blurred, and the characteristic high spin charge-transfer band has appeared at 640 nm. The native and substrate-bound spectra are isosbestic at 406, 454, 556, and 586 nm. No type I compound has been found capable of inducing a conversion of P-4501,~~ from low to high spin greater than 5040% (42). The spectrum shown in Fig. 1 represents the greatest change possible with this compound since, with a K<! of about 2 p ~ , 50 p~ phenylcyclohexane saturates the enzyme. Conversely, by the addition of 1-butanol to ferric P-4501,~~, it is possible to induce a conversion from the native, high spin spectrum to a spectrum strongly resembling that of low spin P - 4 5 0 ~ ~ 2 a s shown in Fig. 3. The 393 nm Soret band has been replaced by one at 416 nm, with an isosbestic point of 406 nm between them. A weak peak near 360 nm has appeared while the 645-nm charge-transfer band has disappeared, and distinct a- and P-bands have been induced a t 567 and 533 nm, respectively. Thus, it is possible to mimic the spectrum of ferric P-4501.~~ by ligation of 1-butanol to ferric P-450LM4. In an experiment not shown, such an effect of 1-butanol could also be observed with P-4501.~~ which had first been converted to the high spin state. Thus, in the presence of phenylcyclohexane (35 p ~ ) and dilauroylglyceryl 3-phosphorylcholine (80 p ~ ) , P - 4 5 0 ~ ~ is about 40% high spin (A418/A393 = 1.34) and the addition of 1-pentanol (final concentration, 46 mM) to this solution caused a reversion to nearly 100% low spin (A4,~/A:x9:, = 2.54).

cm").

Imidazole.

To model possible native ligands other than alcohol or water, the series of compounds shown in Table 111 was chosen. Each of these compounds, when coordinated at the iron sixth position of P-450, should provide a coordination sphere similar to that existing when the corresponding native ligand candidates are present. The spectra resulting from exposure of ferric P - 4 5 0 ~ ~ ~ or P"htjOLM4 to these compounds are indicated in Tables I and IV. With those compounds shown in Table I, it was possible to achieve complete or nearly complete conversion to an alternate spectral species, while those in Table IV produced only a partial change (50% or less) to an alternate spectrum. With p-cresol, hexanoic acid, and benzamide, as with 1-butanol, the spectrum of P -450r .~~ was converted to one resembling that of P-450LMZ (ie. a reverse type I spectral change). In fact, all compounds with a coordinating oxygen atom in the molecule gave this type of spectral change. This list includes alcohols, an ether, ketones, a phenol, carboxylic acids, a hydroxamic acid, and an amide. The amide probably coordinates through its oxygen rather than its nitrogen. The reverse type I change is also observed in the presence of poly(ethy1eneoxy) nonionic detergents such as Renex 690 or Triton N-101. The oxygen-coordinating compounds gave no change with P - 4 5 0 ~ ~ ~ except when phospholipid was present, In that case, the small shift to high spin induced by the phospholipid was reversed by the oxygen compound.

The nitrogenous compounds, 1-benzylimidazole and l-octylamine, gave spectra distinctly different from those of the oxygen compounds (Table I). The spectrum of the coordination complex of 1-benzylimidazole with ferric P - 4 5 0 ~ ~ ~ shows peaks at 359,423, and 540 nm with a shoulder at 570 nm (Fig. 4). The corresponding complex with 1-octylamine, reported in part earlier (39), is similar. The shift from the native spectrum to the 423-nm Soret spectrum represents the conversion of

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WAVELENGTH lnml

FIG. 3. Absorption spectrum of 1-butanol complex P-4501.~4.

of P'450LM4. Ferric P-4501.~~ (3.3 p ~ ) was dissolved in the standard buffer but containing 0.25 M 1-butanol.

-. , Native, ferric P - 4 5 0 1 . ~ ~ (3.3 p ~ ) ; - - -, ferric, 1-butanol complex

TABLE I11 Models for P-450" native ligand

Potential native ligands Model liaand

Oxygen Water, serine, and threonine 1-Butanol Tyrosine p-Cresol Aspartic and glutamic acid Hexanoic acid Asparagine and glutamine Benzamide

Nitrogen Histidine Lysine

1-Benzylimidazole 1-Octylamine

Sulfur Cysteine 1-Octane thiol Methionine Pentamethylene sulfide


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TABLE IV Compounds producing incomplete spectral changes with ferric

P - 4 5 0 ~ ~

Spectral change chrome Compound Cyto-

P-450 Concen- Appar- tration ent K t

Type I LM2 LM2 LM2 LM2 LM4

Reverse type I LM4

LM4 LM4 LM4 LM4 LM4 LM4 LM4 LM4 LM4

No change LM4 LM2 LM2 LM4 LM4

Phenycyclohexane t-Butylperbenzoate Triphenylphosphine p-Nitrocumene 2,4-Lutidine Primary, secondary al-

cohols Acetone Tetrahydrofuran Hexanoic acid m-Chlorobenzoic acid Cyclohexanone p-Cresol Benzohydroxamic acid Benzamide Hydrocinnamonitrile Dimethyl sulfoxide NaN02 Hydrocinnamonitrile p-Nitrocumene I-Octane thiol

rnM

0.05 0.002 0.4 0.4 0.01 0.4

20 2

2300 IO00 500 250 40 40 20

210 10 10 60 50"

8" 10

280 100 100 10 2

160 p~ phospholipid.

l-BllIrn

. . . . . . . . , . . . . . .

~ 16

I?

1 1 I I I I 1 350 400 450 500 550 600 650

WAVELENGTH lnml

FIG. 4. Absorption spectrum of 1-benzylimidazole complexes of P-450~~. -, Native, ferric P - 4 5 0 ~ ~ 2 (3.8 pM); - - -, ferric, 1-benzylimidazole complex of P - 4 5 0 ~ ~ ~ . 1-Benzylimidazole, final concentration 50 p ~ , was added to the native protein. . . . . , The ferric complex was made anaerobic and then photoreduced as described under "Materials and Methods."

one low spin form to a different low spin form with a changed coordination sphere about iron. This conversion is the basis of the so-called type I1 difference spectrum (41). Essentially the same spectrum may also be induced with P " k j o L M 4 by addition of 1-benzylimidazole. Both imidazole and l-methylimidazole elicit this spectrum as well, with only slight differences, and for a given ligand, the spectra of the complexes with p-4501,~~ and P-45oLM4 are difficult to distinguish. Within the series of imidazole derivatives, the apparent K d for the complex with P-4501.~ diminishes as the compound becomes more hydrophobic, and a given derivative binds considerably more tightly to P-4501,~2 than to P-4501,~~. The P-4501.~~ 1- benzylimidazole complex, when reduced, exhibited a spectrum (Table I1 and Fig. 4) which had a Soret maximum at 421 nm and which was isosbestic with the ferric 1-benzylimidazole complex at 413 and 442 nm. A small hump was present at 450 nm with variable intensity. Exposure of the reduced complex to air resulted in immediate oxidation back to the original ferric complex (A,,, a t 423 nm) within 30 s, showing the

reversibility of the reduction reaction of this derivative. Ex- posure of the reduced complex to carbon monoxide generated the expected 450 nm band, but with about 20% P-420 also evident.

The third possible type of ligand is that coordinating through a sulfur atom, of which there are two types: thiol (cysteine) and thioether (methionine). Accordingly, the spectra of the complexes of ferric P-4501.~~ with 1-octane thiol and with pentamethylene sulfide (thiacyclohexane) were determined (Table I and Fig. 5). As in the results of Ruf et al. (20) with P-450,,,, a hyperporphyrin spectrum was observed with 1-octane thiol, the split Soret bands appearing a t 379 and 469 nm. The hyperspectrum probably is a result of Ionization of the thiol to give a bis-thiolate complex. At the solubility limit of the thiol, a small peak remained at 419 nm, which may represent either residual, uncomplexed P-450LM2 or coordinated thiol rather than thiolate. The visible region showed a broad band centered around 550 nm. Because of the incomplete development of the spectrum, molar absorptivities of these bands were not assigned. The thiol failed to react with P-4501,~s at the concentration achievable in the aqueous solvent, probably reflecting the general trend for ligands to bind less tightly to P-4501,~~ than to P-4501.~~. Pentamethylene sulfide bound to the ferric forms of each of the P-450 isozymes, giving essentially the same spectrum with both (Table I and Fig. 5). Surprisingly, the spectrum was very similar to that observed with the amine and imidazole derivatives. Neither the thiol nor thioether-induced spectrum resembled that of native, low spin P-450.

Since some of the reagents were employed at rather high concentration, especially 1-butanol (0.25 M), it was possible that the spectral changes observed were the result of disruption of the tertiary or quaternary structure of the protein due to changes in the bulk properties of the aqueous solvent. Ligation of the compound at the sixth coordination position, as presumed in these experiments, ought to be reversible. The classical method of demonstrating reversible complex forma- tion is the observation of the dissociation of the complex upon dilution. By this criterion, the binding of 1-butanol to P - 4 5 0 ~ ~ ~ was found to be completely reversible. The spectrum of a sample containing P-4501" (9.5 p ~ ) and 1-butanol (0.25 M) was recorded, and then the sample was diluted to one-tenth the original concentration with buffer (Fig. 6). The spectrum of the diluted sample was very nearly that of uncomplexed P- 4 5 0 ~ ~ ~ (A,,, 393 nm). A second sample was allowed to stand in the presence of 0.25 M 1-butanol for 3 h a t 25 "C before

I I I 1 I 350 400 450 500 550 600 650

WAVELENGTH (nml

FIG. 5. Absorption spectra of complexes of P-45Om~ with 1- octane thiol and with pentamethylene sulfide. -, Native, ferric P - 4 5 0 ~ ~ ~ (3.9 p ~ ) ; - - -, ferric, 1-octane thiol complex. Native P-4501.~2 (3.9 p ~ ) was dissolved in the standard buffer but containing 1 mM 1-octane thiol. . . . a , Ferric, pentamethylene sulfide complex. Native P - 4 5 0 ~ ~ ~ (3.9 p ~ ) was dissolved in the standard buffer but containing 1.6 mM pentamethylene sulfide.


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I I I I I 1 400 450

WAVELENGTH (nm)

FIG. 6. Reversibility of 1-butanol binding to P-450Lmz. -, Ferric, 1-butanol complex of P - 4 5 0 1 . ~ ~ . The sample contained P - 4 5 0 ~ ~ ~ (9.5 PM) dissolved in the standard buffer but containing 0.25 M 1- butanol. Full scale is 1 .0 absorbance unit. - - -, The 1-butanol complex was diluted 10-fold with standard buffer not containing I-butanol. Full scale is 0.10 absorbance unit. . . . . , The diluted sample was made anaerobic under carbon monoxide and reduced with sodium dithionite. Full scale is 0.10 absorbance unit.

dilution and conversion to the ferrous carbonyl derivative. At that time, less than 10% P-420 was determined to be present. In another demonstration of reversibility, a solution of P- 4 5 0 ~ ~ ~ (4 p ~ ) was titrated to predominantly the low spin form with 1-butanol. This solution was concentrated by ultrafiltration to about Yi the original volume and then diluted with buffer back to the original volume. A significant return to the high spin form occurred (from 39% high spin to 68% high spin) due to the dilution of the ligand, as judged by the absorption spectrum. The solution was again concentrated and again diluted to the original volume, and an additional conversion to high spin was observed (from 68-83%). The hemeprotein in this solution was then reduced with sodium dithionite in the presence of carbon monoxide to reveal that virtually no P-420 had been formed.

The coordination of 1-benzylimidazole was similarly dem- onstrated to be reversible by dilution. In this case, a mixture of P - 4 5 0 1 , ~ ~ (9.5 p ~ ) and 1-benzylimidazole (85 p ~ ) was diluted to one-tenth the original concentration (Fig. 7). The spectrum of the solution reverted to that of native P - 4 5 0 ~ ~ ~ . Further- more, the ferrous carbonyl derivative of the diluted protein contained only 13% P-420, establishing that the active site structure was not irreversibly altered by coordination of the imidazole derivative. The binding of 1-benzylimidazole to P- 450r.M2 was sufficiently strong in the presence of phospholipid to allow a titrimetric estimation of the number of molecules bound per molecule of enzyme (Fig. 8). The intersection of the line connecting the origin and the first point with the horizontal line representing the maximal absorbance change occurs at 5.7 p ~ , Since the concentration of P - 4 5 0 1 , ~ ~ in the

sample was 4.5 PM, the apparent number of molecules bound per enzyme molecule is 1.3. When correction is made for the fraction of 1-benzylimidazole which is protonated at pH 7.4 (pK, = 6.68), the value of n becomes 1.1, near the value of 1.0 expected for the ligand exchange reaction. The apparent K d of the complex calculated from these data is 0.90 p ~ . If the imidazole derivatives and the alcohols are binding as sixth ligands to iron, then an alcohol should competitively inhibit

w 0 z U K

8 m U

FIG. 7

7 400 450

WAVELENGTH l n m ~ '. Reversibility of 1-benzylimidazole binding to P-

sample contained P - 4 5 0 1 ~ (8.7 PM) and I-benzylimidazole (85 pM) in the standard buffer. At this ligand concentration, the cytochrome is not completely complexed. Full scale is 1.0 absorbance unit. - - -, The 1-benzylimidazole was diluted 10-fold with standard buffer :,ut containing 1-benzylimidazole. Full scale is 0.10 absorbance unit. . . . ., The diluted sample was made anaerobic under carbon monoxide and reduced with sodium dithionite. Full scale is 0.10 absorbance unit.

4501.~4. ~ , Ferric, 1-benzylimidazole complex of P - 4 5 0 ~ ~ . The

[I-EA Im], Total, JJM

FIG. 8. Titration of P-450~~2 with 1-benzylimidazole. Aliquots of I-benzylimidazole were added to a solution of P - 4 5 0 1 . ~ (4.5 @" and the spectrum recorded after each addition. The sum of the absolute magnitudes of the absorbance changes at 410 and 432 nm relative to the spectrum of the native enzyme was determined and plotted for each concentration of ligand. The small arrow indicates the intersection of the extrapolation of the initial slope with the maximal absorbance change.


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the binding of an imidazole. This was found to be the case with both P-45oLM2 and P"t50LM4. Titration experiments with 1-benzylimidazole similar to the one shown in Fig. 8 were performed in the presence and absence of an alcohol. Phos- pholipid was not added to the enzyme solutions, and pentanol rather than butanol was used since the enzymes could be saturated at lower concentrations of pentanol than butanol, thereby avoiding possible changes in bulk solvent properties. The apparent K d for t h e P - 4 5 0 ~ ~ 2 complex with l-benzylimidazole increased from 7.3 p~ in the absence of pentanol to 47 p~ in the presence of 30 mM pentanol. The apparent Kd for the P-450LM4 complex with 1-benzylimidazole increased from 2.5 to 10 p~ when about 60 mM pentanol was present. Another preparation of P - 4 5 0 ~ ~ ~ showed an increase in apparent Kd from 3.4-23 p~ under similar conditions. These increases in apparent K d range from 4- to 7-fold and appear to be consistent with competition of different ligands for the same binding site.

Several other types of ligands, which do not correspond to potential native ligands, were also examined. Some of these have been extensively studied with other hemeproteins and offer the opportunity to probe the ligand-binding site of cytochrome P-450 and compare its behavior to a protein such as myoglobin. Five nitrogenous compounds in this category are shown in Table I. 0-Benzylhydroxylamine produces the same type I1 spectrum as shown by octylamine. Picoline (4-meth- ylpyridine) shifts the ferric Soret maximum of P-45oLM to longer wavelength (422 nm), but not as much as do the imidazoles and amines. The &-region is similar to that with the amines. Lutidine (2,4-dimethylpyridine) has a sterically hindered nitrogen and is unable to coordinate to iron. This compound was found to bind simply as a hydrophobic substrate, inducing the typical type I shift to the high spin state. Another hindered base, 2-phenylimidazole, induced a shift of the P-450LM4 spectrum to one similar to that of the native P- 450r.M2. The spectrum of the complex with P-4501.~~ was readily distinguished from the native spectrum in that the absorption maxima were red shifted by 1-2 nm with little change in peak shape. Since the apparent Kd for the 2-phenylimidazole interaction did not change with successive recrys- tallizations, it is unlikely that the spectral change is due to an oxygen-coordinating impurity. It would appear that weak coordination through the hindered nitrogen produces a ligand field nearly the same as that produced by a coordinating alcohol. Nitric oxide (NO) binds strongly to both P-450r.M2 and P - 4 5 0 ~ , ~ ~ in the ferric state, producing a large shift of the Soret band to 433 nm and eliciting a new peak at 359 nm, along with distinctive a- and P-peaks at 575 and 543 nm. Isosbestic points were at 390 and 426 nm. This spectrum is similar to that reported for the nitric oxide complex of P- 450,.,,, by O'Keefe et al. (12). Upon exposure of the ferric complex to sodium dithionite, a transient absorbance near 440 nm was observed, followed by rapid loss of absorbance a t all wavelengths in the Soret region.

Benzyl isocyanide forms a complex with ferric P-45oLM2 showing peaks at 362,430, and 547 nm (Table I). The spectrum is isosbestic with native P-4501.M.) at 378, 425, 516, 535, 562, 585, and 618 nm. The ferric isocyanide complex may be reduced with sodium dithionite to yield a ferrous derivative with two bands in the Soret region (Table 11). With P-450LM2, the ratio A4:~2/A4rr6 was 0.7, while with P-450r.M4, the corresponding ratio A4a:3/A4,1 was 1.9 at pH 7.4. In each case, the peak near 430 nm may represent the isocyanide complex with P-420, formed during the reduction step (43). In that case, the 456 nm peak probably is the low energy component of a hyperspectrum (23).

Organic phosphines are good ligands to transition metals, and Ruf et al. (20) have reported the ligation of diethylphen-

I I I I 1 I I

100 I P - 4 5 0 ~ y ~ 20

+ CN-

.16

1 1 I 1 1 350 400 450 500 550 600 650

WAVELENGTH inmi

FIG. 9. Absorption spectrum of cyanide complex of P-4501.~~. -, Native, ferric P-4501.~2 (4.2 p ~ ) ; - - -, ferric cyanide complex of P-4501,~~. Potassium cyanide, fins1 total concentration of cyanide (CN- and HCN) 50 mM, was added to the native enzyme.

ylphosphine to P-450,,,. This compound was found to bind strongly to P-450LM2 and P-&Ol,M4 as well (Tables 1 and 11). The spectrum of the ferric complex was a typical hyperporphyrin type with peaks at 377, 452, and 560 nm. In contrast, the less basic, more hindered triphenylphosphine induced a type 1 spectrum (Table IV). Surprisingly, P - 4 2 0 ~ ~ ~ gave almost the same spectrum with diethylphenylphosphine as P- 4501.~2 (Table I), and the spectrum with hemin chloride was also similar (376, 450, and 552 nm). These results suggested that the strongly coordinating phosphine might have completely removed the heme from the protein. However, gel filtration of the mixture of P-450l.Mr and diethylphenylphosphine on Sephadex G-25 showed that the reaction of the phosphine with P-450 was completely reversible. The spectrum of the eluted protein was that of native P-450LM2, and the ferrous carbonyl derivative was estimated to contain 5% or less P-420. A second gel filtration experiment was conducted in which the G-25 column was equilibrated with the phosphine before application of the P-4501.~~ phosphine complex. The spectrum of the eluted protein was the same as that of the applied sample, indicating that the protein carried the heme with it. No heme was retarded by the column. Clearly, the heme is not dissociated from the protein by this ligand, although nonspecific binding would not be distinguished from the native mode of binding. However, major disruption of the heme-binding site appears unlikely in view of the maintenance of integrity of the P-450 following removal of the phosphine by gel filtration. Thus, we conclude that diethylphenylphosphine binds to P-450 as a sixth ligand without drastically disturbing the native heme binding site and, in particular, with retention of the proximal thiolate ligand. Why, then, do P-420 and hemin yield complexes with this phosphine with such similar absorption spectra to the P-450 complexes? Both the P-420 and hemin complexes are probably bis-phosphine compounds. Ruf et al. (20) showed that bis-thiolate and thiolate phosphine complexes with ferric protoporphyrin IX dimethyl ester had very similar hyperspectra, with some over- lap of wavelengths, depending on the particular thiolate used. It seems reasonable, then, that a bis-phosphine hemin complex would have a spectrum that could not easily be distinguished from the spectrum of the P-450 phosphine complex.

Four other compounds were found to serve as ligands for P- 4 5 0 ~ ~ : potassium cyanide, potassium thiocyanate, sodium azide, and potassium fluoride. The spectra produced by these small anionic ligands are reported here for the first time. The ferric P - 4 5 0 ~ ~ 2 cyanide complex has a spectrum (Table I, Fig. 9) qualitatively similar to that of myoglobin cyanide (36) except that with P-450, all peaks are red shifted and the near


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ultraviolet peak has an intensity approaching that of the Soret. The cyanide-induced spectrum is isosbestic with native P-4501,~~ a t 380, 430, 518, 543, 564, and 583 nm. Potassium thiocyanate has been used for the denaturation of P-450 (11). At a concentration of 1.1 M KSCN, both P-450LM2 and P- 4 5 0 ~ ~ ~ were denatured to the P-420 derivatives. At lower concentrations of KSCN, the conversion to P-420 did not occur appreciably, and the binding of thiocyanate to the iron could be studied. In the presence of 0.92 M KSCN, P-45oLM2 formed the ferric thiocyanate complex with a decreased Soret band at 419 nm (Table I). The spectrum of this complex was isosbestic with native, ferric P-450LM2 at 383,430, 559, and 583 nm. A double reciprocal plot of absorbance changes versus concentration indicated an apparent K d of 0.35 M. Sodium azide elicited a spectrum with ferric P - 4 5 0 ~ ~ ~ (Table I) which was qualitatively similar to that of the imidazole complex. This spectrum was isosbestic with native, ferric P“k50LM2 at 398,429,561, and 581 nm. A double reciprocal analysis yielded an apparent Kd of 0.2 M, explaining why it is possible to inhibit catalase with a concentration of sodium azide (e.g. i mM) which is without effect on cytochrome P-450 (44). The reported spectrum, taken at 1.6 M azide, was stable; however, at 2 M azide, rapid precipitation of the protein occurred.

Reaction of P-4501.~2 with potassium fluoridc was also observed, as judged by spectral changes. Identical spectral changes were observed with sodium fluoride, but the lower solubility of the sodium salt severely limited the extent of their development. A double reciprocal analysis indicated an apparent K d of 1.8 M for the ferric P-450LM2 F- complex. However, no KF concentrations higher than 1.5 M were used since protein precipitation set in at about 1.8 M. This apparent Kd is very high with respect to that of other hemeprotein fluoride complexes. For example, the fluoride complex of cytochrome oxidase exhibits a K d of only 25 mM (45). The presence of KF at concentrations of 1.5 M or below induced a difference spectrum (fluoride complex minus native, ferric cytochrome) with a maximum at 384 nm, a minimum at 421 nm, and isosbestic points a t 406 and 437 nm. This spectrum was similar to a type I difference spectrum (41) but was distinct in the position of the maximum, the second isosbestic point, the presence of substantial positive absorbance above 437 nm, and in the exact curve shape. Still, the difference spectrum suggested conversion of low spin native P“i50LM2 to a high spin form, but not the same high spin form induced by type I compounds. Using a difference millimolar absorptivity of 126 mM” cm” for a type I change (46) combined with the extrapolated maximal absorbance change in the fluoride difference spectrum obtained from the double reciprocal plot, one may estimate that only 20% of the hemes in the sample can interact with fluoride or, more likely, that all of the hemes can interact, but that the resulting ferric fluoride complex is 80% low spin. In contrast, ferric myoglobin fluoride is 96% high spin (47).

DISCUSSION

This work has attempted to characterize the sixth, or distal, ligand to heme iron in native, ferric P-45oLMZ. The experimen- tal approach was to ligate the iron with a series of exogenous compounds which can mimic particular endogenous ligand candidates. Comparison of the spectra of the resulting coordination complexes with the spectrum of the native cytochrome served as a means of judging the ligands similar to or different from the native ligand. In the process, a broad exploration of the reactions of the iron coordination sphere was carried out. Particularly useful was the opportunity to add ligands to P-45oLM4, which is without a distal ligand, and match the spectrum of native P-450LM2. The spectra of P-

4 5 0 ~ ~ 2 and P - 4 5 0 ~ ~ 4 were found to be nearly identical when the ligation states are the same, both as the ferric and ferrous derivatives. Thus, when an added ligand induces in P-450LM4 a Spectrum Similar to that of native P-4WLMZ we assume that the two ligation modes are similar. Fortunately, the spectra induced by the ligands fell into just a few groups, as will be discussed in more detail below. The spectra obtained with the ligands in Table I11 were of three types: a “nitrogen” type, shown by benzylimidazole, octylamine, and pentamethylene sulfide; a “hyper” type, shown only by 1-octane thiol; and an “oxygen” type, shown by 1-butanol, p-cresol, hexanoic acid, and benzamide. The “oxygen” type ligands induced little or no change in the spectrum of P - 4 5 0 ~ ~ ~ , but changed the spectrum of P - 4 5 0 ~ ~ 4 t o one closely resembling that of native P-45oLMZ. However, the other two types induce spectra with P-450LM2 or P-45oLM4 which resemble the native spectra of neither of these cytochromes. Thus, of the ligands in Table 111, only the “oxygen” type reproduces the native spectrum of P - 4 5 0 ~ ~ 2 upon coordination to P-450LM4. Since all of these compounds contain a coordinating oxygen atom, we surmise that the native sixth ligand of P - 4 5 0 ~ ~ ~ coordinates through an oxygen atom, rather than a nitrogen or sulfur atom. Fur- thermore, inasmuch as all known forms of native, low spin cytochrome P-450 have virtually identical optical and EPR properties, we can extrapolate this conclusion to all of these forms.

This conclusion rests on three assumptions. First, P - 4 5 0 ~ ~ 2 and P“i5oLM4 exhibit closely similar spectra when their ligation modes are essentially the same. Second, the added substances do not induce gross disruptions at the heme locus. Third, the added substances directly coordinate as ligands to iron. We note the following facts and observations as justifi- cation for these assumptions. With respect to the first assumption, the native low spin forms of all known types of cytochrome P-450 have essentially superimposable spectra. Also, these enzymes all bind substrates to produce high spin ferric forms with similar spectra. In addition, in this work, a particular ligand did, indeed, induce the same spectrum whether it reacted with P-45oLMZ or P-450LM4. With respect to the second assumption, the binding of an exogenous, some- times large, molecule in the heme binding region will, of course, produce alterations of local structure. These can in- clude displacement of the native distal ligand, movement of the iron in or out of the mean heme plane with consequent doming or buckling of the porphyrin macrocycle, and movement of the peptide backbone and R-groups. However, with most of the ligands used in this study, no severe, irreversible changes were observed. With 1-butanol, 1-benzylimidazole, and diethylphenylphosphine, evidence for the reversibility of the reaction of the ligand with P-45oLM was presented. The existence of isosbestic points was taken as an indication of the lack of irreversible changes as well, although this could be misleading. On the other hand, some ligands or conditions were clearly disruptive, as for instance the binding of NO to ferrous P - 4 5 0 ~ ~ or the reduction of p-4501.M in the presence of high concentrations of alcohol. In these cases, several signal phenomena were usually present, including unstable spectra, conversion of P-450 to P-420, and protein precipitation. No conclusions were drawn about ligation of particular compounds when there was an indication of disruption.

The third assumption, that these added compounds directly ligate to heme iron, may be the most critical. Fortunately, several circumstances add to the veracity of this assumption. EPR (43,48) and Mossbauer spectroscopy (49), both of which probe the state of the iron atom itself, show profound changes in the presence of such ligands, suggesting direct coordination to iron. Griffin et al. (50) and Pirnvitz et al. (51), using


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nitroxide-labeled substrates, have shown direct coordination of pyridine and isocyanide derivatives, respectively. Also, some of the types of compounds are known to interfere with each other’s binding in a competitive manner (43, 52), and in the present study, the binding of pentanol was shown to compete with the binding of 1-benzylimidazole. In addition, the binding of small entities such as CO, NO, 0 2 , CN-, N3-, and NCS- can only reasonably occur at the heme iron since they do not show hydrophobic binding and no other transition metals are present in cytochrome P-450. This fact shows that the iron is indeed susceptible to ligand replacement reactions. Finally, if these compounds are not bound at the iron, then there must exist a second site which transmits the binding event to the heme. This hypothetical allosteric site would have to affect the heme differently depending on whether the added compound is a hydrocarbon, alcohol, amine, or thiol, etc. In contrast, one can readily explain all of the spectral changes observed by postulating ligand replacement reactions at iron. In fact, ligation of all of these types of compounds is well established in several other hemeproteins (36,40,47) and in isolated iron porphyrins (19, 20).

In many respects, cytochrome P-450 is similar in general spectral behavior to other, more typical, hemeproteins. How- ever, one unique feature of cytochrome P-450 is the conversion of a hexacoordinate low spin state to a five-coordinate high spin state upon reduction (53). This fact is, in itself, suggestive of oxygen distal ligation since in all known cases in which the ferric state of a hemeprotein has an imidazole ligand, that ligand is retained in the ferrous derivative. For instance, cytochrome bs has two imidazoles in both states and cytochrome c has an imidazole and a methionine in both states. Conversely, myoglobin remains high spin in both ferric and ferrous states since it has no distal imidazole. In fact, we expect a nitrogenous ligand to bind more strongly to the ferrous state than to the ferric state of cytochrome P-450, as has been shown for the pyridine complex of P-450,,, (54). Ferrous cytochrome P-450 becomes low spin when an exogenous sixth ligand is provided, as has been shown to be the case for CO (49) and NO (12). This should be true of nitrogenous ligands such as primary amines and imidazole derivatives as well. Thus, the fact that ferrous cytochrome P-450 is high spin argues against the availability of a protein-donated imidazole to serve as a distal ligand.

After consideration of the spectra induced by all of the various compounds examined, we have grouped the ligands into four classes based on their molecular features and their spectral behavior with ferric cytochrome P-450. Type 0 ligands produce spectra with a barely resolved near ultraviolet band at 361-362 nm, a Soret band a t 417-418 nm, and resolved, nearly equivalent ,R- and a-bands at 533-535 nm and 567-569 nm, respectively. These ligands coordinate through a hard nucleophile such as an oxygen atom. Fluoride may be of this class. Type N ligands produce spectra with a well-resolved near ultraviolet band in the range 359-366 nm, a Soret band occurring at 419-426 nm, and an unresolved a$ pair, with the ,&band a t 538-541 nm and the a-band as a shoulder near 570 nm. These ligands coordinate through a softer nucleophile such as nitrogen or sulfur. The sterically hindered ligand 2- phenylimidazole exhibits behavior intermediate between that of the type 0 and that of the type N ligands. Type H ligands produce hyperspectra, characterized by equally intense near ultraviolet and Soret bands (“split Soret”) falling at 377-379 nm and 452-469 nm, respectively, and a single but very broad band in the 550- to 560-nm region. Type P ligands may form r-bonds with iron through back bonding of the iron t2g d- orbitals to the ligand r*-antibonding orbitals. The spectra produced feature a strong, well-resolved near ultraviolet peak

at 359-365 nm, a Soret band at 430-436 nm, and a single broad band in the visible region at 543-559 nm. With nitric oxide, a weak a-band is partially resolved. The four classes are not as evident in the spectra of ferrous P-450 derivatives. Thus, type N and type H ligands are not readily grouped by their spectra with ferrous P-450. Type 0 ligands do not bind to the ferrous state. However, type P ligands all produce hyperspectra with ferrous P-450 similar to that of ferrous carbonyl cytochrome P-450. Since O2 is a diatomic r-bonded ligand, we would expect it to also produce a hyperspectrum with ferrous P-450. However, if there were net transfer of electron density from iron to oxygen so that the ferrous dioxygen complex resembled ferric superoxide, then one might expect ferrous dioxygen cytochrome P-450 to exhibit a spectrum similar to that produced by type 0 or type N ligands with ferric cytochrome P-450. The spectrum of ferrous dioxygen P-450,,, has been reported (52, 55) and, in fact, does show similarity to the ferric type 0 and type N ligand spectra. Sharrock et al. (49) have previously suggested a ferric superoxide configuration based on Mossbauer parameters.

The ligands in Table I may be placed into the four classes as follows: type 0, 1-butanol; type N , imidazole, l-methylimidazole, 1 - benzylimidazole, 1 -octylamine, 0- benzylhydroxylamine, picoline, sodium azide, pentamethylene sulfide, and potassium thiocyanate; type H , 1-octane thiol and diethylphenylphosphine; and type P , potassium cyanide, benzyl isocyanide, and nitric oxide. Each of the ligand types shown produces a low spin complex upon coordination to cytochrome P-450. These ligand types should not be confused with the spectral types (type I, type 11, and reverse type I) introduced by Schenkman et al. (41,56), but they are related. Thus, type I spectra are induced by removal of the sixth ligand to produce a pentacoordinate high spin iron; type I1 spectra are induced by type N ligands; and reverse type I spectra are induced by type 0 ligands.

The present investigation complements earlier studies on the nature of the iron sixth ligand in cytochrome P-450. NMR (30,33) studies of P-450,,, have revealed a rapidly exchanging proton which is strongly coupled to paramagnetic iron and which is approximately 2.6 8, away from the iron. Electron nuclear double resonance spectroscopy (34) has shown the presence of exchangeable protons coupled to the iron spin of P-450,,,. One of the strengths of electron nuclear double resonance is the reliable assignment of distances between a metal center and coupled protons, using a principle entirely different from that on which NMR estimates of such distances rests. Nonetheless, the estimate of the iron-proton distance from electron nuclear double resonance (2.6 to 2.9 8,) is in excellent agreement with the estimate from NMR. Ligands which were suggested to be compatible with these results are water, tyrosine, serine, threonine, lysine, arginine, asparagine, and glutamine (33). Histidine, cysteine, aspartic acid, and glutamic acid were not compatible. We have found that the native-type low spin spectrum can be induced with ligands corresponding to water, tyrosine, serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. Histidine, lysine, cysteine, and methionine could be eliminated from further consideration. Arginine was not specifically tested. It would undoubtedly produce a spectrum like that of octylamine if it were to coordinate, but its high pK, would appear to effectively make it unavailable for coordination to iron. Fur- thermore, we believe that the amides, asparagine and glutamine, are in reality eliminated by the NMR results. The amides are expected to coordinate to iron through the carbonyl oxygen atom (57), not through the amido nitrogen. For instance, peptides bind to zinc in carboxypeptidase A through the carbonyl (58). Measurements on molecular models indi-


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cate an iron-proton distance of 4.2 A when an amide coordinates through the carbonyl oxygen. Analogous measurements on models of the water or alcohol complexes indicate an iron- proton distance of 2.7 A. Thus, this work narrows the list of possible sixth ligands to water, serine, threonine, and tyrosine. The differentiation of water from the other three candidates could potentially be realized by the observation of quadrupo- lar broadening by “0 water of the EPR absorbances of iron (26).

On the other hand, recently published pulsed EPR studies on low spin ferric P-450,,, indicate nuclear modulation of the iron spin echo envelope by a nonporphyrinic nitrogen atom (29). The authors presented arguments that this nitrogen atom was the outer, noncoordinating nitrogen of an imidazole ligand. Nuclear modulation by the directly coordinated nitrogen atom was not observed. However, the nuclear modulation pattern from a putative imidazole-heme-mercaptoethanol model complex was not a good match to that obtained from P-450,.,,,, although the agreement between the model and the mercaptoethanol adduct of myoglobin was excellent. We have considered several alternative ligand configurations which might allow weak electron-nuclear coupling with a remote nitrogen atom and yet be consistent with our conclusion that the directly ligated atom is oxygen. A carbonyl-coordinated amide might show the observed nuclear modulation, but as pointed out above, the distance between iron and the exchangeable protons in such a complex is not in accord with the NMR and electron nuclear double resonance measurements on P-450,,,. That the modulation pattern observed represents a minority population of imidazole-bound iron (e.g. P-420) does not seem likely due to the depth of modulation exhibited. Finally, we consider the model suggested by Ristau et al. (32) in which an iron-coordinated water molecule (or another hydroxyl ligand such as serine) is hydrogen bonded to an imidazole, as in hemoglobin. This structure could be consistent with all data, provided that weak iron-nitrogen coupling may occur across the hydrogen bond, The fact that coupling occurs between metal and protons of second shell solvent water molecules in aqueous lanthanide samples pro- vides some support for such hydrogen bond-transmitted coupling (59).

Finally, since there is now a body of evidence implicating an oxygen sixth ligand in cytochrome P-450, we may ask whether oxygen is a reasonable choice. Certainly an oxygen ligand would be readily displaced from the ferric ion by stronger nucleophiles such as amines, imidazoles, and cyanide, etc. Also, an oxygen ligand would probably not coordinate to ferrous iron, thus allowing this state of cytochrome P-450 to be pentacoordinate high spin. This property is important since it opens a coordination position in ferrous heme for dioxygen ligation, However, oxygen ligands are not normally ascribed sufficient field strength to force the low spin configuration in transition metal complexes. Thus, one may question whether a hexacoordinate heme with an oxygen trans to thiolate could reasonably be low spin. We believe that such a set of ligands could indeed produce the low spin state for the following reasons. Most importantly, the spin state of a heme complex is largely a matter of the geometry of the complex.2 Thus, low spin hemes are hexacoordinate, while high spin hemes are pentacoordinate. Exceptions are now known to this generali- zation, but they occur only when both axial ligands are of the weak field type, typically neutral oxygen donors (60). This general rule arises because much of the splitting of the eR and

’ Although the coordination polyhedron of the cytochrome P-450 low spin iron does not possess octahedral (0~~) symmetry and the true symmetry is no higher than C,, , the Oh model nonetheless has utility in this discussion.

orbital sets is due to the ligand field exerted by the four pyrrole nitrogens of the porphyrin itself on the iron d.+ -.,;I orbital. The presence of ligands along the axis of the+ orbital will likewise raise its energy away from that of the fZp, orbitals, giving the familiar split of the eg and tZg orbital sets. If one or both of the axial ligands is of moderate to strong field strength, then the magnitude of the energetic splitting will require the pairing of d-electrons into the lower energy t2, orbitals. Only if both axial ligands are of the weak field type can electrons significantly populate the eg orbitals. Since no hemeproteins have yet been described in which both axial ligands are the neutral oxygen type, it is likely that the rule stated above remains valid for hemeproteins. In the particular case of cytochrome P-450, the presence of the proximal thiolate anion, which is charged and is capable of d-d back bonding, should be sufficient to favor the low spin state in a hexacoordinate configuration regardless of the nature of the trans ligand.

On the other hand, when the complex is pentacoordinate, the iron moves out of the heme mean plane, and the pyrrole ligands are no longer directly on the axes of the d , ~ -?? orbital lobes; the clear splitting of the ep and tZg orbital sets is lost. Also, with one less ligand, the total field strength felt by the pentacoordinate iron is decreased, such that all orbitals are closer together in energy. As a result, electrons may populate all five d-orbitals and the complex is high spin. Acid metmy- oglobin might be used as a counterexample to the preceding arguments since it apparently has a water molecule as a sixth ligand and is high spin. However, x-ray analysis shows that the iron is 0.3 A below the heme plane (36). The Fe-0 distance is 2.3 A, which is too long to represent a coordinate covalent bond. Thus, the water molecule is only a loosely associated outer sphere ligand.

One should also keep in mind in this discussion that ferric cytochrome P-450 is not completely low spin. The equilibriclm constant between high and low spin forms is only about 12-15 in favor of the latter (32, 53), corresponding to a free energy difference of about 1.5 kcal/mol. With such a small difference, ferric cytochrome P-450 is close to the crossover point between high and low spin, so that postulation of a relatively weak oxygen ligand becomes even more reasonable. Interestingly, P - 4 5 0 1 , ~ ~ ~ as isolated, is mainly high spin (39), emphasizing that a strong ligand such as an imidazole is not available. That this form of P-4501,~ is high spin has been ascribed to the presence of 1 mol of tightly bound substrate ( i e . 3-methyl- cholanthrene) per mol of heme (61). However, other preparations, which contained as little as 0.2 mol of 3-methylchol- anthrene/mol of heme, were nonetheless completely high spin (62). There was no evidence for bound substrate in the P- 4501,~~ preparations used in the present work, and we believe this protein is intrinsically high spin.

The apoprotein may act to control the heme spin state. Phenomena through which such control may be exerted in- clude the movement and availability of the axial ligands and possibly deformations of the porphyrin macrocycle attending its binding to the protein. In ferric cytochrome P-450, the protein may tend to force the low spin hexacoordinate configuration by “pushing” the iron into the plane through its ligands. In fact, such protein control of the spin state in cytochrome P-450 would be more effective if the sixth ligand were also a protein-donated one rather than a water molecule. If the cytochrome P-450 apoprotein is designed to maintain the low spin state of the hemin, there may be a functional reason. Upon binding of a hydrophobic substrate, P-4501.~2 is partially converted to the high spin form. The molecular mechanism for this conversion may be the opening up of the active site and consequent pulling away of a protein-donated sixth ligand from the iron caused by the insertion of the organic molecule into the hydrophobic binding region. As


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shown by Sligar and co-workers (53, 63), an increase in the high spin content of P - 4 5 0 ~ ~ raises the apparent reduction potential so that those cytochrome P-450 molecules to which substrate is bound may be "switched on" for catalysis of substrate hydroxylation by virtue of being more readily re- ducible. Conversely, those molecules with no bound substrate remain low spin and low potential so that few reducing equiv- alents from NAD(P)H would be directed toward them. Thus, an oxygen ligand could allow the protein to maintain the resting low spin state until a hydroxylatable substrate was present. At that point, an oxygen ligand, unlike an imidazole ligand, would be easily removed so that the impending reduction and dioxygen-binding steps could commence.

Acknowledgments-We are grateful to Dr. Robert C. Blake I1 for performing the gel filtration, to Dr. Daniel D. Oprian for determining the pK, of 1-benzylimidazole, and to both for useful ideas and critical comments. We also thank Dr. J. A. Peterson for insights into heme spin states, Dr. Stephen G. Sligar for discussions, and Dr. J. A. Fee for advice on the interpretation of pulsed EPR results. Finally, we gratefully acknowledge Dr. John H. Dawson, who communicated results of a similar study prior to its submission for publication.

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R E White and M J Coonbonding atom of the axial ligand trans to thiolate as oxygen.

Heme ligand replacement reactions of cytochrome P-450. Characterization of the

1982, 257:3073-3083.J. Biol. Chem.

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