Eur. J. Biochem. 71, 63-76 (1976)

Five-Coordinate Iron-Porphyrin as a Model for the Active Site of Hemoproteins Characterization and Coordinating Properties Michel MOMENTEAU, Michel ROUGEE, and Bernard LOOCK Labordtoire de Biophysique du Museum National d'Histoire Naturelle, Paris (Received July 6/September 15, 1976)

Preparation of iron(II1)-deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been performed using the mixed carboxylic/ carbonic-acid-anhydride method. This compound, which is very soluble in various organic solvents, can be considered as a prosthetic group model for the active site of five-coordinate hemoproteins. In the oxidized state a basic, a neutral or an acid form can be isolated. The basic and acid forms are monomeric at all concentrations. The neutral form is found dimeric in concentrated solutions while it is monomeric at low concentration. The coordination state of iron in these various species is investigated. The neutral form reacts with ligands, such as nitrogenous organic bases, leading to six-coordinate well-known hemichromes which exhibit low-spin electron spin resonance (ESR) spectra. The reaction of anionic ligands, such as F-, CN-, NOT and N;, with the neutral form model leads to unsymmetrical six-coordinate complexes whose optical and ESR spectra are similar to those of synthetic deuteromyoglobin. In benzene, toluene or dichloromethane solutions iron(I1)-deuteroporphyrin 6(7)-methyl ester, 7(6)-histidine methyl ester), obtained from ferric forms by heterogeneous reduction with aqueous dithionite, exhibits an optical spectrum characteristic of a five-coordinate high-spin ferrous complex. At low temperature important spectral modifications are observed indicating a dimeric association. At room temperature it binds one nitrogenous base molecule leading to the well-known hemochrome. It reacts also with carbon monoxide with a very high affinity constant (4.5 x lo8 M-'), comparable to that of the isolated human hemoglobin CI and fi chains, but much higher than the values reported for other various models, suggesting that the side-chain length bearing the fifth ligand may have an important influence upon the reactivity of the sixth position of the iron ion. At low temperature in toluene or dichloromethane, this compound reversibly binds oxygen without oxidation of the iron ion while oxidation occurs at room temperature. The significance of these results is discussed in relation with the local environment, the electronic nature of the base and the immobilization of the heme group in hemoproteins.

Histidine plays an important role in the hemeprotein interactions [1,2]in the process of oxygen binding in hemoglobin [ 3 ] and related myoglobin [4,5].X-ray structures of these hemoproteins have revealed that the prosthetic group, a protoheme, is bound to only one histidine residue (known as proximal histidine). A second histidine residue (known as distal histidine) is located near the iron ion on the Ahhreviutions. Fe"'Dtp(OMe,His-OMe), Fe"Dtp(OMe,HisOMe), oxidized and reduced iron-deuteroporphyrin 6(7)-methyl ester 7(6)-(histidine methyl ester) ; His-OMe, histidine methyl ester; Im : imidazole ; MeIm : 2-methylimidazole; PyCN, 4-cyanopyridine; CHO-Cys(Ph3C)-His-OMe, N-a-formyl-S-(triphenylmethy1)cysteine-histidine methyl ester; Me-S-Pro, methylpropyl sulfide; ESR, electron spin resonance.

opposite face of the porphyrin ring [6]. Both histidine residues are believed to participate in oxygen binding. Because oxygen binding studies in isolated hernes show that a rapid oxidation of Fe" into Fe"' takes place at room temperature, various investigators have attempted to simulate the local geometry and electronic environment of hemoglobin and myoglobin active sites in simple heme compounds in an effort to impede iron ion oxidation. A route to the synthesis of a hemoglobin model involves l-(2-phenylethyl)imidazole-heme imbedded in a polystyrene backbone [7]. The heme iron is considered to be complexed by imidazole only in one axial position. Lautsch [8], Sano et al. [9] and Momenteau and Loock [lo] have described model

64

heme derivatives containing ligands covalently bound to the vinyl side-chains of protoheme via thioether linkages, as in cytochrome c. Another possible route is to link the ligand group to the propionic acid sidechain of the heme molecule. Thus, recently, Warme and Hager [ll], Chang and Traylor [12] and Castro [13] have described hemes with an imidazole covalently bound in a position allowing it to act as an axial ligand towards the iron ion. However, these models are almost insoluble in organic solvents. The 3-(1imidazo1yl)propyl-pyrroheme of Chang and Traylor [12] binds oxygen without undergoing oxidation at low temperatures, but oxidation occurs at room temperature. A synthetic iron-porphyrin with exceptional steric hindrance on one side of the ring (a picketfence porphyrin) has been prepared by Collman et al. [14], and its complex with l-methylimidazole reversibly binds oxygen at room temperature without Fe” oxidation for numerous cycles. All these five-coordinate iron-porphyrin complexes have been synthesized to investigate the influence of the protein chain upon the reactivity of the active site principally in the fixation of oxygen. These studies describe qualitatively their autoxidation and general chemistry, but few quantitative investigations have been reported on their physicochemical properties. In this paper we report the preparation of irondeuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) (Table 1, compound 8) in which the histidine amino group is covalently bound to one propionic acid side-chain of the porphyrin ring and the imidazole group of histidine is able to be coordinated to one of the axial coordination sites of iron. This compound is very soluble in organic solvents (up to 0.01 M) in which the non-polar heme environment is comparable to the hydrophobic crevice of the protein. We describe quantitative coordinating reactions of this model in the oxidized and reduced states towards nitrogenous bases, inorganic anions, carbon monoxide and oxygen as the sixth ligand. The various derivatives of the model in the ferric state have been characterized by their ESR spectra. Besides, we compare our results with those obtained with hemes, other model systems and myoglobin. MATERIALS A N D METHODS Materials

Deuterohemin (Table 1, compound 1) was prepared by a previously described procedure involving treatment of protohemin with resorcinol [15]. Dimethylformamide was distilled under reduced pressure of nitrogen and stored on a 0.4-nm molecular sieve. All solvents of the purest available grade were purchased from Baker and Merck companies. Silica

Five-Coordinate Iron-Porphyrin

gel was purchased from Baker Co. L-Histidine methyl ester, methylpropyl sulfide, imidazole and 2-methylimidazole were purchased from Fluka company. CHO-Cys(Ph3C)-His-OMe was prepared as described earlier [16]. Prediluted CO in nitrogen, grade LJ in volume) was specially supplied by Air(1.08 x Liquide. Iron ( I I I )-deuteroporphyrin 6 (7 )-Methyl Ester, 7(6)-(HistidineMethyl Ester) (Compound 8 ) 0.19 ml(2 mmol) of ethylchloroformate was added to a solution of 1.128 g (2 mmol) of deuterohemin (compound 1) and 0.28 ml (2 mmol) of triethylamine in 35 rnl of dimethylformamide at -5 “C. After stirring for 2 h at - 5 “C, 0.464 g (2 mmol) of L-histidine methyl ester dihydrochloride and 0.56 ml(4nimol) of triethylamine in 20 ml of dimethylformamide were added. After standing overnight at room temperature some insoluble triethylammonium chloride was filtered off and the crude product precipitated upon addition of ether. 200mg of the crude product was dissolved in a mixture of butan-l-ol/dcetic acidlwater (4/3/1, by vol.) and applied on a 2.5 x 34-cm silica gel column. Three fractions A - C were successively eluted with the same solvent. After solvent evaporation under reduced pressure, each fraction was dissolved in chloroform/ methanol (1/1) and identified by its absorption spectra as: A, deuterohemin (compound 1) and C, deuterohemin 6,7-bis(histidine methyl ester) (compound 9). Fraction B will be identified from its physicochemical properties as iron(II1)-deuteroporphyrin 6(7)-(histidine methyl ester) (compound 6). Yields were respectively: 0.201 g, 0.547 g and 0.318 g (16%, 35% and 17 % based on the starting material). 0.547 g of compound (6) was dissolved in 150 ml of absolute methanol with 1 % HCl. After standing 3 h at room temperature the solution was poured into chloroform (500 ml). The organic solution was successively washed with water, aqueous sodium bicarbonate and then water, dried over sodium sulfate and evaporated to dryness. The solid material was dissolved in a minimal volume of a petroleum-ether/ chloroform/methanol mixture (0.5/1/0.3, by vol.) and applied on a silica gel column previously treated with the same mixture. One minor component was removed from the column and spectrophotometrically identified as iron(I1I)-deuteroporphyrin dimethyi ester (compound 3). A second component was eluted with a chloroform/methanol mixture (1/0.3, v/v) and identified by infrared spectrometry as methoxo-iron(II1)deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) (compound 7). This solution was evaporated to dryness. The residue was dissolved in chloroform and shaken vigorously with a NaC1-saturated aqueous solution acidified at pH 5 with HC1. The organic

M. Momenteau, M. Rougee, and B. Loock

Table 1. Structures Compounds

1 2 3 4 c

6 7 8 9 10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26 27 28 29 30

31

of

65

model in ouidi:ed and reduced .stater and derivntivc~

Iron State

R' (R')

R" (R')

X

X'

oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized oxidized

OH OCH, OCH, OCH,

OH OCH, OCH,

-

c1-

OCH, OCH, His-OMe His-OMe His-OMe Hjs-OMe His-OMe His-OMe His-OMe Hjs-OMe His-OMe His-OMe His-OMe His-OMe His-OMe

Im Im ? Hi-OMe His-OMe Hjs-OMe Hjs-OMe Hj-OMe His-OMe His-OMe FHis-OMe CNHjs-OMe

OCH,c11m1+ ? OCH,C1His-OMe]' Im]' MeIm]' OHFF-1CNCN-1NO2-

Hjs-OMe

N,-

reduced reduced reduced reduced reduced reduced reduced reduced reduced reduced reduced reduced reduced

[OCH, OH OCH, OCH, [His-OMe [OCH, [OCH, OCH, OCH, LOCH, OCH, [OCHB OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH, OCH,

layer was dried over anhydrous sodium sulfate. (Cl-)Fe"'Dtp(OMe,His-OMe) (compound 8) was precipitated upon addition of petroleum ether. Yield : 0.392 g (26 % based on the starting deuterohemin). Thin-layer chromatography (silica gel 60 thinlayer chromatography plates, Merck) showed that

-

? ?

?

c1-

His-OMe His-OMe His-OMe His-OMe His-OMe His-OMe His-OMe I Hi-OMe His-OMe OCH,

His-OMe His-OMe Im His-OMe CHO-Cys(Ph,C)His-OMe His-OMe MeIm His-OMe PyCN 4ICN PyCN His-OMe Me-S-Pro His-OMe CO O2 His-OMe -

-

OCH, OCH,

Im MeIm

-

OCH,

PyCN

-

-

the compound (8) moved as a single spot with RFvalue of 0.51 in the solvent system petroleum-ether/chloroform/methanol (0.35/1/0.3, by vol.). Under the same conditions deuterohemin dimethyl ester (compound 2) and deuterohemin 6,7-bis(histidine methyl ester) (compound 9) had RF values of 0.64 and 0.38 respectively.

66

Five-Coordinate Iron-Porphyrin

The compound (8) is characterized by its infrared spectrum and its elementary analysis. The carbonyl frequenciesare: 1735 cm-' and 1432 cm-' (COester); 1665 cm-', 1530 cm-' and 752 cm-' (amide I, I1 and V). Elementary analysis for C38H39N705FeCl was as follows. Calcd: C, 59.66; H, 5.14; N, 12.82; C1,4.63. Found: C, 59.97; H, 5.2; N, 12.92; C1,4.27. Preparation of Iron (II)-deuteroporphyrin 6(7)-Methyl Ester 7(6)-(Histidine Methyl Ester) (Compound 19) Solutions

The reduction of Fe"'Dtp(OMe,His-OMe) (compound 8) in benzene, toluene or dichloromethane by aqueous sodium dithionite, as well as the procedure used to transfer under inert atmosphere the Fe"Dtp(OMe,His-OMe) (compound 19) solution into the optical cells for spectrophotometric titration (pathlengths of 1 mm and 10 mm for the Soret and visible regions respectively) have been performed as previously described for deuteroheme dimethyl ester (compound 28) [ 16,171. Rigorously anhydrous benzene and toluene were obtained according to the method of Szwarc et al. [18], using polystyryl anion, an active polymer, and kept under vacuum using a glass apparatus similar to the one described elsewhere [19]. Solutions of compound (19) in these rigorously anhydrous solvents were obtained by vacuum distillation of the solvent into cells containing a known amount of solid (compound 19) resulting from the evaporation todryness(lO-'torr, 1.332 mPa, 70 "C) of a water-saturated benzene solu-

1

tion of the reduced model (in such conditions the very stable dipyridinehemochrome quantitatively loses its two pyridines [20]). Methods

Optical spectra in the Soret and visible regions were recorded using a Cary 15 or a Beckman DKU spectrophotometer. Temperature was regulated at 25 f 0.1 "C. The spectrophotometric titrations in chloroform or benzene with various ligands under inert atmosphere have been performed as described elsewhere [16,17,21]. The dimerization of compound (8) has been spectrophotometrically studied as described by West and Pierce [23]. The ESR spectra were recorded on a Varian 4502 X band spectrometer. The magnetic field strength was calculated from the nuclear resonance frequency of a proton probe located close to the cavity (Varian F8 A magnetometer). This frequency was determined with a Hewlett Packard 5245 L frequency counter. The microwave frequency was measured with a calibrated wavemeter. Samples were frozen in liquid Nz. RESULTS Spectral Studies on Fe"' Dtp(OMe,His-OMe) : Coordination State of'Iron

The optical spectra of the water-soluble hemins are strongly pH and concentration dependent, and different states of ligandation are well characterised. Accordingly, shaking chloroform or dichloromethane

.o

c(

0.5

0

350

400

500

600 X inm)

Fig. 1, Optical uhsorption spectra of0.08 m M (opticulputh, visible I cm, Sorer I mm) Fe"'Dtp(OMe,His-OMe) in chloroform. ('basic' (compound 12), (- - -) form I1 'neutral' (compound 8) and (-.-.-) form 111 'acidic' (compound 8 protonated). ( + + 0 ) represent the Soret absorption spectra of 1.6 pM form I, form 11 and form 111 respectively (optical path 5 cm)

~

) Form I

+ +,

--,

M . Momenteau, M. Rougee, and B. Loock

61

solutions of Fe'"Dtp(OMe,His-OMe) with KC1-saturated aqueous solutions leads to different species, depending on the pH of the aqueous phase and of the concentration of the organic solution, each of them being characterized by different optical spectra. a) At pH 12, using a 0.08 mM Fe"'Dtp(OMe,HisOMe) organic solution, we observed that the optical spectrum presents a visible band at 585 nm and a Soret band at 396 nm (Fig. 1 form I 'basic'). This spectrum is concentration independent. b) Upon lowering the pH of the aqueous phase, important spectral modifications are observed : two new bands appear at 632nm and 532nm, with a shoulder at about 505 nm. The Soret band shifts from 396nm to about 402nm, with a shoulder at about 380 nm (Fig. 1, form I1 'neutral'). The spectral evolution from form I to I1 is achieved at pH 5-6. However, the ratio of the absorbances at 402nm and 380 nm and, to a lesser extent, the features of the visible spectrum, depend on the concentration of the solution, as will be shown later. c) Lowering further the pH from 5 to about 1.5 leads to a third type of spectrum (Fig. 1, form 111 'acid'), in which the charge-transfer band at 632 nm is sharpened, whereas the band at 402 nm decreases and the band at 380 nm increases. The latter spectrum is similar to that of deuterohemin dimethyl ester in the same solvent, except for a minor modification of the 532-505-nm band ratio (0.98 and 1.05 for the model and deuterohemin respectively). It is also comparable to the absorption spectrum of the 3-(1imidazoly1)propyl-pyrrohemin synthesized by Chang and Traylor [12] and Castro [13]. No major spectral modifications with concentration are observed. Addition of tetrabutylammonium hydroxide, a strong base, to chloroform solutions of forms I1 and I11 restores the form I, which can thus be identified

I (basic) compound (1 2)

hemin dimethyl ester (compound 2). This last spectral modification can be assigned to the displacement of the protonated histidine from the fifth coordination position of the iron ion. This is also probably the case for the 'acidic' form 111, but a little doubt exists because of the slight differences observed in the visible part of the spectra. The situation is more complicated in the form I1 case, to which we have devoted most of our attention. Dilution experiments show that Beer's law is not obeyed when the concentration is lowered from 0.02 M to 7 pM (visible region, optical paths 0.1 mm to 5 cm) and from 2 mM to 0.6 pM (Soret region, optical paths 0.1 mm to 5 cm). The spectral modifications are specially important in the Soret region, where the 404-nm to 382-nm absorption ratio decreases from 1.25 to 0.86. The same evolution is observed at a fixed model concentration upon increasing the temperature, or upon addition of a large excess of tetraethylammonium chloride. These dilution and temperature effects suggest that the evolution from the 'diluted' form I1 to the 'concentrated' form I1 reflects an exothermic dimerization process with intermolecular binding of one histidine of one model molecule to the free sixth coordination site of a second one (Scheme 1). The dimerization equilibrium constant at 25 "C is found to be KD = 1 x lo5 M-'. This equilibrium has been studied at different fixed model molecule concentrations as a function of the temperature. The enthalpy change, calculated according to the Van't Hoff equation between 18 "C and 58 " C , is found to be AH = - 79 kJ mol-'. From this value and AG = - RTlogK, one deduces the entropy change AS = - 170 J mol-' K-'. The following Scheme (1) summarizes at the molecular level the structure of the different observed forms :

I1 (neutral monomeric) compound (8)

as the six-coordinate species having the histidine strongly bound in the fifth and a hydroxyl ion bound in the sixth coordination positions of the iron ion (compound 12). On the other hand, addition of acetic acid or bubbling gaseous hydrochloric acid in chloroform solutions of forms I and I1 leads to a species whose absorption spectrum is comparable to that of deutero-

I1 (neutral dimeric)

111 (acid) compound (8) protonated

Spectral studies on Fe"Dtp(OMe,His-OMe) (Compound 19) : Coordination State of Iron The reduction of (C1-) Fe"'Dtp(OMe,His-OMe) (compound 8) in benzene, toluene or dichloromethane gives a red compound, stable in absence of oxygen. The absorption spectrum at 53 pM of compound (19)

68

Five-Coordinate Iron-Porphyrin

Table 2. Absorption maxima of Fe'"Dtp(OMe,His-OMe) and Fe"Dtp(OMe,His-OMe), and their derivatives For comparison the absorption maxima of bare deuteroheme and six-coordinate deuterohemochromes are reported. Absorption maxima are given in nm, molecular absorption coefficients (between parentheses) in ml mol-' . cm-' Compound

Absorption maxima

(8)

in

Soret region

Solvent visible region

nm (ml mot-' cm-') 12 8 (concentrated) 8 (diluted) 8 (protonated) 10 11 13b 15b 17 18 19 20 21 22 23 24 25 26 27 ' 28 Fe"Dtp(Me-S-Pro)z CO(1m)Fe"Dtp a

396 380 380

402 402

380 403 398 392 409 405 408 414 (108) 412 (195) 412 (177) 413 (142) 405 405 (124) 410 (148) 409 (205) 403 387 (65) 407 (68) 418 (125) 410 (265)

470 505 505

585 532

632

532 527 531

632

480

590 533 529 532 562 610 520 ( 8.7) 546 (10.7) 518 (15.8) 547 (20.5) 518 (14.9) 547 (19.7) 518 (11.4) 548 (16.7) 514 (15.4) 544 (19.8) 710 ( 5.0) 514 (15.9) 544 (24.3) 630 (10.0) 519 (17.6) 549 (19.5) 528 (12.2) 555 ( 8.7) 530 562 526 ( 9.7) 559 (11.5) 519 (17.1) 549 (16.2) 528 (12.1) 556 ( 8.8)

chloroform chloroform chloroform chloroform chloroform chloroform dimethylformamide dimethylformamide dimethylformamide dimethylformamide benzene benzene benzene benzene benzene benzene methylpropylsulfide benzene toluene benzene methylpropylsulfide benzene

Not determined (see text). 0.2-0.7 mM in added salt At -60 "C.

in benzene is shown in Fig.2. The concentration is determined assuming that the hemochromes obtained from compound (19) and deuteroheme dimethyl ester with imidazole, which have identical spectra, also have the same ~ 5 4 7= 20500 [22]. The same spectrum is obtained (a) whatever form of Fe"'Dtp(OMe,HisOMe) is undergoing reduction; (b) in rigorously anhydrous benzene or toluene. It is characterized by one broad visible band centered at 546nm, with a shoulder near 520 nm, and a Soret band at 414 nm, with a weak shoulder near 420 nm. This spectrum is clearly distinct from both that of bare deuteroheme dimethyl ester (compound 28) [16] and that of typical bis(histidine) deuterohemochrome in the same solvent. However, it is quite similar to those obtained with the well-identified five-coordinate species, deoxydeuteromyoglobin [24] and mono-(2-methylimidazole) deuteroheme dimethyl ester (compound 30), except for an inversion of the absorbance at 423nm and the shoulder near 415 nm [25] is the case of the latter. Lowering the temperature of a toluene solution of compound (19) leads to a hemochrome-like spectrum at -60 "C, with an increase of the absorbance at 547 nm, the appearance of a new (B) band at 518 nm, and a slight blue shift of the Soret band to 408 nm accompanied by an increase of its absorbance. The

same spectral modifications are observed in rigorously anhydrous toluene. This observation rules out the binding of water as sixth ligand, both at room and low temperature, and suggests, as for compound (8), a dimer formation by intermolecular exothermic coordination of the histidine of one Fe"Dtp(OMe,HisOMe) (compound 19) molecule to the sixth coordination position of another one, according to the following Scheme (2) :

His compound (19)

-Fe

Tciluene freezes at - 95 " C , before the dimerizaztion of compound (19) is completely achieved. The spectrum recorded at -90 "C is clearly distinct from the hemochrome one recorded at the same temperature. It is also different from the expected dimer spectrum, obtained using a 131 mixture of bare heme and hemochrome, and can be accounted for by an approximate 60 % conversion of compound (19) into dimer.

M. Momenteau, M. Rougte, and B. Loock

69

2

. 1.0

w Lo

7

21 ~

0.5

0

0

400

600

500

x (nm) ; deuteroheme dimethyl ester (compound 28) ( Fig.2. Optical spectra of Fe"Dtp(OMe,His-OMe) (compound 19) (-) (CHO-Cys(Ph3C)-His-OMe)Fe"Dtp(OMe,His-OMe) (compound 21) (- . - . -) in benzene. Concentration : 53 pM The same spectral modifications are detected at 25 "C, but only with very concentrated solutions of compound (19). The optical spectrum recorded in the visible region with the minimal optical path of 0.1 mm (concentration of about 6 mM) gives only 30% conversion into dimer, as estimated from the comparison with bare deuteroheme and hemochrome spectra recorded in the same conditions. At 25 "C we can deduce an upper limit of the dimerization constant, K D = lo2 M-', which is much lower than that of compound (8). The approximately 0.04 mM solutions (Cl-)Fe"'Dtp(OMe)2 compound (2)

compound (4)

Action of Nitrogenous Organic Bases Titration of very diluted (i.e. monomer) solution of compound (8) by imidazole in chloroform progressively leads to a species whose absorption spectrum is similar to that of six-coordinate bis-(imidazo1e)deuterohemin dimethyl ester (compound 5). As discussed earlier in detail [25] quantitative analysis of the spectral modifications shows that this ligandation process obeys a 131 stoichiometry. The affinity con~ M-'. stant was found to be K = 2 . 3 lo5 Titration of more concentrated solutions does not fit this simple scheme because monomer and dimer are present in solutions. Moreover, corrections for bound imidazole become important. The mathe-

-)

and

matical analysis of this system taking into account the monomer-dimer equilibrium, gives an affinity constant K = 1.8 x 10' M-', in good agreement with the one obtained in diluted solution. For comparison, titration of deuterohemin dimethyl ester (compound 2) in chloroform by imidazole occurs in a single step, involving the fixation of two imidazole molecules (stoichiometry 1/2) with an overall ~ M-2. The intermediate speaffinity plpz = 1 . 4 lo7 cies, compound (4), in the following sequential Scheme (3)

% (Cl-) (Im)Fel"Dtp(OMe)z

used for the titration experiments can thus be essentially considered as monomer solutions.

-

Im

[(Im)~Fe1"Dtp(OMe)2]+C1compound (5)

(3)

is not observable as shown by the presence of isosbestic points and the linearity of the l/A-Ao versus l/[1ml2 plot [16]. Titration of the very diluted form I1 (compound 8) solutions by 2-methylimidazole progressively leads to a species having a hemichrome-like spectrum, but with a less intense Soret band at 398 nm. Quantitative analysis of the spectral modifications does not obey the simple law characterizing a 131 stoichiometry. Half-saturation occurs at about 0.1 mM in 2-methylimidazole. Fe"Dtp(OMe,His-OMe) (compound 19) in benzene gives with imidazole and CHO-Cys(Ph3C)-HisOMe, six-coordinate low-spin hemochromes, e.g. compounds (20,21), well identified by their optical spectra (Fig. 2). The titrations satisfy all the requirements (isosbestic points, linear plots) indicating the binding of one ligand molecule, with affinity constants

Five-Coordinate Iron-Porphyrin

70

but with a less intense jband. Hence the a / j ratio is higher than in symmetrical hemochromes (Fig. 3). The titration is consistent with the binding of one 2-methylimidazole molecule to compound (19) with an affinity constant K = 1.8 x lo3 M-l. 4-Cyanopyridine has also been investigated because the characteristic features of the spectral modifications in the red allow us to analyse two well-distinct steps during the titration. In the first one a 4-cyanopyridine molecule binds to compound (19) leading to a species (compound 23) with a hemochrome absorption spectrum, and with a high affinity constant K = 1 x 105 M - I . Further modifications of the intermediate spectrum are observed in the red, only when the concentration of 4-cyanopyridine is greatly increased. Only minor changes of the intensity of the a and j bands are recorded, leading to a species (compound 24) whose spectrum is identical to that of the blue bis(4-cyanopyridine)deuteroheme dimethyl ester obtained from deuteroheme dimethyl ester and 4-cyanopyridine [22], which also has a broad absorption band centered at 630 nm. Analysis of the spectral modifications at this wavelength gives for the second step, ascribed to the replacement of histidine methyl ester by a second 4-cyanopyridine molecule, an affinity constant K = 35 M-'.

K = 1.1 x 105 M-' and K = 3 x lo4 M - ' respectively. These values are comparable to the second affinity constant (and much higher than the first one) measured by the titration of bare deuteroheme dimethyl ester (compound 28) with these ligands in the same solvent (6.8 x 104 M-l for imidazole [25] and 3.7 x 104 M-' for CHO-Cys(Ph3C)-His-OMe). Addition of 2-methylimidazole to compound (19) in benzene gives rise to a hemochrome-like spectrum, Table 3. $$ninify

constants of Fe"Dtp(OMe,His-OMej (compound

19). deuteroheme dimethyl esfer (compound 28), various model

porphyrins and some hemoproteins for diiferent ligands at 25 "C

K

Compound

References

~

M-' 19 + 19 + 19 + 19 + 19 +

19 < imidazole CHO-Cys(Ph3C)-His-OMe 2-methylimidazole 4-cyanopyridine

1 x lo2 1.1 x lo5 3 x 104 i . 8 x lo3 I x105 35 19 + methylpropyl sulfide 8 4.5 x lo8 19 + CO 28 + imidazole 4.5 x lo3 6 . 8 lo4 ~ 28 + 2-methylimidazole 1.2 x lo4 28 + CHO-Cys(Ph3C)-His-OMe 2.5 x lo3 3 . 7 lo4 ~ 4.8 x 10' 29 + CO 5.6 x lo7 31 CO CO 4.4 x 105 Pyridine-mesoheme 1 x lo6 Imidazole-pyrroheme CO Horse myoglobin CO 2.2 x 107 Human hemoglobin 2" chain + CO 3 x108 4.8 x lo8 pSHchain + CO 3.1 x lo8 Chironomus hemoglobin + CO

+

+

+

(1) (2)

(1) (2) (I) (2)

this work this work this work this work this work this work this work this work ~251 ~ 5 1 1251 this work this work

PI1

Action of Various Anions on (C1-)Fel"Dtp(OMe,His-OMe) (Compound 8 ) in Dimethylformamide

[211 [311 1321 1331

+

The action of various anions on (compound 8) has been studied in dimethylformamide because salts are rather insoluble in chloroform. Important spectral modifications are observed, thus revealing

1341

1341 P51

0 1

400

500

600

X inm)

Fig.3. Optical spectra of Fe"Dtp(OMe,His-OMe) (compound 19) (-) in benzene. Concentration 53 pM

and (MeIm)Fe"Dtp(OMe,His-OMe) (compound 22) ( - - -)

M. Momenteau, M. Rougee, and B. Loock

71

1.0

7

0.5

0 300

400

modifications of the ligation state of the iron ion. Unfortunately, quantitative studies are impossible because dissociation constants of the added salts in dimethylformamide are unknown. With tetraethylammonium fluoride and potassium cyanide, two well-distinguishable steps occur, as shown by the presence of two series of well-defined isosbestic points. The first one, observed for relatively small anion concentrations (0.2-0.7 mM in added salt) gives species (compounds 13 and 15) whose absorption spectra are identical to those of the corresponding deuteromyoglobin derivatives [26], in which chloride ion is replaced by fluoride or cyanide ion. Excess anion (0.7 - 5 mM) converts them into difluoride and dicyanide complexes (compounds 14 and 16), as revealed by their absorption spectra, which are identical to those obtained with deuterohemin dimethyl ester [27] (Fig.4). Only the first step, e.g. the formation of compounds (17) and (18) is observed by addition of sodium azide and sodium nitrite to a dimethylformamide solution of our model. The optical properties of the azide and nitrite derivatives are identical to those of the deuteromyoglobin derivatives [26]. Action of Carbon Monoxide and Oxygen on Fe"Dtp(OMe,His-OMe) (Compound 19)

Compound (19) in benzene with the minimal pressure of CO that can be obtained with our experi-

500

600

7

mental set-up (prediluted CO, 108 parts/106 further diluted 50-fold with argon, leading to p C 0 = 2 x atm. (0.2 Pa) and free [CO] = 13 nM) gives more than 80 % conversion to the monocarbonyl complex (compound 26), so that only the upper end of the titration curve can be observed. The optical spectrum of compound (26) is comparable to that of mono-(imidazo1e)mono-(carbony1)deuteroheme dimethyl ester (Fig. 5). The calculated affinity constant K = 4.5 x lo8 M-' is comparable to that of mono(imidazo1e)deuteroheme dimethyl ester (compound 29) for CO (4.8 x lo8 M-' P I ) . Admission of oxygen to a compound (19) toluene solution at -60 "C immediately gives a new species (compound 27) whose absorption spectrum (Fig. 5) is characterized by two visible bands at 562 nm and 530 nm, and a Soret band at 403 nm. This oxygenation at low temperature is fully reversible, and bubbling argon restores the initial spectrum. Warming the oxygen complex to room temperature results in the irreversible Fe"Dtp(OMe,His-OMe) oxidation.

ESR Spectra of (Cl- )Fe'"Dtp (OMe,His-OMe) (Compound 8 ) ESR spectra have been obtained in frozen chloroform and dimethylformamide at 77 K. This compound exhibits both high-spin (g = 6) and low-spin ( g = 2.91; 2.28; 1.55) signals (Fig.6). As shown earlier [16,27] in the same solvents and at the same

Five-Coordinate Iron-Porphyrin

12

01

500

400

x

600

inm)

Fig.5. Opfical spectra of Fe"Dfp(OMe,His-OMe) (compound 19) (---) and ( C O ) Fe"Dtp(OMe,His,OMe) (compound 26) (at room temperature in benzene. Optical spectrum of ( 0 2 ) Fe"Dtp(OMe,His-OMe) (compound 27) (-.-.-) at -60 "C in toluene

-

-1

Table 4.Principal values of the g tensor f o r the model and its derivatives at 77 Kin dimethylformamide For comparison, the principal values of the g tensor for some hemichromes and deuteromyoglobin derivatives are given

.-

Compound

g tensor values

12 8" 10 13b 15b 18 5 9 Deuteromyoglobin + azide Deuteromyoglobin + hydroxide

2.51 6 2.91 2.93 6 2.85 2.81 2.93 2.91

Reference

a,

> .-

m

>

I

a, D

c .+ 0

a I

0 yi

L1

m

LT

a W

Magnetic field

Fig. 6. ESR spectrum of (('1.- j Fe"'Dtp(OMe,His-OMe) pound 8 ) f o r m 11 'neutral' (0.01 M ) in chloroform at 77 K

(com-

temperature, deuterohemin dimethyl ester (compound 2) is typically high spin and deuterohemin 6,7-bis(histidine methyl ester) (compound 9) typically low spin. Addition of tetraethylammonium chloride or fluoride to compound (8) results in an increase of the high-spin signal and a decrease of the low-spin

a

2.20 2.28 2.27 2 2.28 2.20 2.27 2.27

1.73 1.55 1.53 2.04 1.90 1.53 1.54

this work this work this work this work this work this work [I61 ~ 7 1

2.84 2.19 1.65

[261

2.48 2.13

[261

1.85

Dimeric form (see text). 0.2-0.7 mM in added salt.

one, whereas addition of organic nitrogenous bases or NT, OH- and CN- gives almost pure low-spin species. The spectral characteristic, affinity constants and ESR g tensor values are reported in Tables 2, 3 and 4 respectively.

M. Momenteau, M. Rougke, and B. Loock

DISCUSSION The method we have developed to obtain a model molecule, soluble in non-polar organic solvents, in which a covalent sequence linked to a propionic acid chain of the deuteroporphyrin ring bears a terminal histidine residue which thus can be coordinated to the fifth coordination site of the iron ion, gives yields similar to that of the sulfuric anhydride method developed by Warme and Hager [ l l ] and others preparations [13]. The use of non-polar solvents is thought to reflect the hydrophobic environment of many five-coordinate hemoproteins. Our model is chromatographically homogeneous and behaves as a unique species, although it is not possible to separate the positional isomers at the 6- 7 positions of deuteroporphyrin ring that must be produced in the coupling reaction. The spectral modifications observed by shaking chloroform solutions of the oxidized molecule with aqueous solutions at various pH values reflect modifications of the close environment of iron ion. The 'basic' form I (compound 12) presents an absorption spectrum similar to that of metmyoglobin in alkaline solution, except for a blue shift of 10- 15 nm of the absorption bands due to the replacement of the hydrogens by vinyl groups at the 2 and 4 substitution positions. In this compound iron ion is coordinated by the histidine methyl ester and a hydroxyl ion. Several lines of evidence indicate that intermolecular associations are present in solutions of form 11, depending on concentration and temperature. Quantitative analysis of the spectral modifications upon dilution are best explained by a dimerization between a monomer and a dimer with a dimerization constant KD = 1 x lo5 M-' at 25 "C, and a relatively large entropic factor A S = - 170 J mol-' K-', as expected for a dimerization process. This dimerization constant is close to, but a little smaller than the affinity constant K = 2.3 x lo5 M-' of the monomer for free imidazole. The 131 stoichiometry indicates, at least, that histidine becomes coordinated during the ligation reaction because only one imidazole is required to transform the monomer into a six-coordinate hemichrome species. Moreover, there is strong evidence that histidine is already coordinated in the monomer. Assuming as a first approximation that the second affinity constant p 2 of (Cl-) (Im)Fe"'Dtp(OMe)2 (compound 4) for imidazole is of the same order of magnitude as the affinity constant K of the model monomer (compound 8) for this ligand, we obtain p1 = i . 4 x 107 M - 1~2 . 3 lo5 ~ M-' = 60 M-l < p z , thus explaining the impossibility to detect the intermediate species during the titration of deuterohemin dimethyl ester (compound 2) by imidazole (Scheme 3). It is highly speculative to correlate the approximate f l 1 value defined above with the intramolecular,

13

dimensionless, concentration-independent binding constant Kin.

His-Fe'm

I c1-

-

4,

-

[I+I c1-

(4)

compound ( 8 )

because this correlation rests upon the ill-defined signification of the local equilibrium concentration of histidine methyl ester near the iron ion. This equilibrium is involved in the dimerization process (Scheme 1). That KD is smaller than K arises from the fact that the removal of histidine methyl ester from one monomer molecule to give the dimer is unfavorable. Low-temperature ESR spectra of frozen concentrated chloroform and dimethylformamide solutions of (Cl-)Fe"'Dtp(OMe,His-OMe) (form 11, Scheme 1) exhibit both high and low-spin signals. As shown above, dimerization is strongly favored at low temperature and in concentrated solution, so that the ESR spectrum reflects the magnetic properties of the dimer. According to Peisach et al. [28], two kinds of ligand environment for hemin iron are revealed, in agreement with the proposed dimer structure, having one low-spin iron coordinated by two strong-field histidines while the second is high-spin and free of strongfield ligands. Consequently ESR cannot tell us whether the monomer, compound (8), is high-spin with an out-of-plane iron or low-spin with an in-plane iron. On the contrary, the above results unambiguously show that in non-coordinating solvents at room temperature, except at high concentration, Fe"Dtp(OMe, His-OMe) (compound 19) is a typical five-coordinate monomer with the histidine methyl ester bound in the fifth axial coordination position of iron. It can thus be considered as a model for the active site of fivecoordinate hemoproteins like myoglobin. According to our own results in rigorously anhydrous solvents, water is not implicated either at room or at low temperature. In pyrroheme amide, another myoglobin-active-site model, Chang and Traylor [I21 have first suggested that the iron ion was probably weakly bound by imidazole in the fifth, and by water in the sixth axial coordination positions. In a subsequent paper [29] they show that their isolated-site model is almost entirely five-coordinate at room temperature, thus excluding the presence of water in the sixth site, even in aqueous solution. However, they do not exclude the possibility that water becomes coordinated at low temperature, as a result of an affinity constant increase. In agreement with our results they report that at low temperature the splitting of the 546-nm band into the so-called CI and p bands also occurs in rigorously anhydrous solvents

14

such as toluene. We feel that the spectral modifications at low temperature, or at room temperature with very concentrated solutions, are best explained by intermolecular, exothermic dimerization (Scheme 2), as observed with the oxidized form. Such a dimerization is not observed with deoxymyoglobin, even at 77 K, because heme-heme interactions are hindered by the protein chain. The dimer formation involves both the histidine methyl ester release from iron and its coordination in the sixth position of the iron of another Fe"Dtp(OMe,His-OMe) (compound 19) molecule. The latter can be qualitatively related to the affinity of compound (19) for externally added imidazole or CHOCys(Ph3C)-His-OMe. Titration experiments show that this is very favorable, as in the case of (CI-)Fe"'Dtp(OMe,His-OMe) (compound 8). Again it is speculative to link the intermolecular, dimensionless, concentration-independent equilibrium constant describingsa hypothetic equilibrium between two monomers having either a bound or an unbound histidine methyl ester, and the first affinity constant of deuteroheme for CHO-Cys(Ph3C)-His-OMe or imidazole. Nevertheless, the very low dimerization constant of compound (19) with respect to compound (8) ( < 1 x lo2 M - ' versus 1 x lo5 M-') makes it possible to conclude that histidine methyl ester is more firmly bound to iron in the reduced than in the oxidized form. This conclusion may be related to our earlier observation that the binding of the first imidazole to deuterohemin dimethyl ester ( K w 60 M-') is much less favorable than is observed with deuteroheme dimethyl ester (3.5 x 103 M-' , [251). Imidazole and CHO-Cys(Ph3C)-His-OMe bind to compound (19) with affinity constants as high as the second affinity constant of bare deuteroheme dimethyl ester for these ligands, which shows that the trans effect exerted by the covalently bound histidine methyl ester towards the reactivity of the sixth coordination site is not restricted with respect to what is observed for monoimidazole or mono-[CHOCys(Ph3C)His-OMe]deuteroheme methyl ester. This means that the covalent linkage between histidine methyl ester and the porphyrin ring does not hinder the in-plane shift of iron upon sixth coordination. Correspondingly the sixth ligand binding strengthens the histidine-iron bound. Although the hemochrome system is more stable than the hemichrome system (the overall affinity constants for the binding of two imidazoles are 3.1 x lo8 M-' and 1 . 4 lo7 ~ M - 2 for deuteroheme dimethyl ester and deuterohemin dimethyl ester respectively), the ratio of the successive affinity constants is lower for deuteroheme dimethyl ester ( z 15, [25]) than for deuterohemin dimethyl ester ( > 4 x lo3), and the relative increase of stability of the six-coordinate with respect to that of the five-coordinate

Five-Coordinate Iron-Porphyrin

species is higher for the oxidized than for the reduced form, in relation to the lower dimerization constant of the latter. Substitution of histidine methyl ester by externally added ligands can be related to the above observations. For Fe"Dtp(OMe,His-OMe) (compound 19), it is only visible with 4-cyanopyridine because the mixed hemochrome (compound 23) obtained in the first step and the symmetrical bis-(4-~yanopyridine)hemochrome (compound 24) have clearly distinct optical spectra in the red. Such a substitution cannot be excluded in the case of added imidazole or CHOCys(Ph3C)-His-OMe, but it is not observed in oxidized or reduced states probably because it is expected to have a little influence, if any, on the optical spectrum. The markedly different behavior of 2-methylimidazole is undoubtedly due to steric effect induced by the methyl group which destabilizes the complex (compound 11) with respect to compound (10). This destabilisation is even more important in the reduced model, with an affinity constant 60-fold lower than the one obtained with imidazole, and has been observed during our earlier studies on the binding properties of reduced deuteroheme dimethyl ester [25]. The optical spectrum of the 2-methylimidazole complex (compound 22) exhibits the characteristic feature of six-coordinate hemochromes, but with a and fl bands less intense than those of others symmetrical or unsymmetrical unhindered hemochromes, even with poor ligands such as methylpropyl sulfide (unpublished results). The existence and position of a Soret band are not characteristic of six-coordinate species, as is the appearance of two a and fl bands. The a/P ratio depends on the nature of the axial ligands, decreasing in the order nitrogenous-nitrogenous (1.30 - 1.40), nitrogenous-sulfide (1.1) and sulfide-sulfide (< 0.95). Both the appearance and the position of well-distinct, sharp and intense a and fl bands seem to be less related to the nature of the ligands and the stability of the hemochromes than to an unambiguous in-plane, low-spin iron. Such a situation cannot be ascertained in the case of the six-coordinate complex with 2-methylimidazole. Quantitative studies of the binding of anions to (C1-)Fe1"Dtp(OMe,His-0Me) (compound 8) are impossible because of the unknown dissociation constants of the salts in dimethylformamide. However, the analogy of the optical and ESR spectra of these derivatives with those of the deuteromyoglobin derivatives [26] shows that the iron environment is the same. The 19Fhyperfine splitting at g = 2 is evidence for a specific hemin-fluoride complex in which the fluoride anion is bound to ferric ion. This splitting is also observed with the myoglobin derivative in which the symmetry of the heme has been altered while remaining attached to the proximal histidine [30]. Addition of excess fluoride converts the latter

15

M . Momentedu, M . Rougee, and €3. Loock

into a high-spin difluoride complex, having an optical spectrum identical to the difluoride derivative of deuterohemin dimethyl ester, and an ESR signal g = 2 split into a triplet [27] indicating histidine replacement by a second fluoride anion. This behavior, also observed with cyanide anion, can be linked to the strong nucleophilicity of these anions. The poor nuccleophilic azide and nitrite anions give only the monocomplexes. Thecompetition between fluoride or cyanide anions and histidine is not observed in the case of metmyoglobin and methemoglobin, either because the proximal face of the heme is unaccessible to externally added ligands, or as a consequence of the constraints exerted by the protein chain upon the heme-histidine bond, or both. Perhaps the most interesting property of Fe"Dtp(OMe,His-OMe) (compound 19) is its very high affinity for CO. Referring to our earlier studies on the competition between CO and imidazole for the sixth position of mono-(imidazo1e)deuteroheme dimethyl ester (compound 29) (Table 3), it can be shown that compound (19) binds CO as firmly as the latter, (as observed above for imidazole). The affinity of the compound (19) for CO is much higher than the values reported by Chang and Traylor [31,32] for their various models bearing a pyridine or imidazole covalently bound to the propionic acid chain of a mesoheme and coordinated to the fifth coordination position of iron. This great decrease (about 1000-fold) cannot be accounted for only by the difference in the electronic properties of imidazole and pyridine. Imidazole is a better o donor and a poorer TC acceptor than pyridine, so that TC back-bonding is more efficient in stabilizing the imidazole-carbonyl with respect to the pyridinecarbonyl complex. However, the increase of stability from pyridine-mesoheme to imidazole-pyrroheme is only 2-fold [12]. Additionally our own results show that the affinity of compounds (19) or (29) for CO is less than 10-fold higher than that of mono-(4-cyanopyridine)deuteroheme dimethyl ester (compound 31). In fact, the models of Chang and Traylor, and our own model differ by the length of the side-chain bearing the fifth ligand, and we believe that the reactivity towards CO is mostly governed by the constraints exerted by the side-chain upon the in-plane shift of iron upon CO bonding. The affinity of compound (19) for CO is 20-fold higher than the affinity of deoxymyoglobin (2.2 x lo7 M-', [33]) and is comparable to the affinity of the most reactive hemoproteins like isolated CI and B chains of human hemoglobin (3.0 x 10' M-' and 4.8 x 10' M-' , [34]) and the single chain Chironomus thummi thummi hemoglobin (3.1 x 10' M-' 1351). With respect to compound (19) and mono-(imidazo1e)deuteroheme dimethyl ester (compound 29), the protein chain of these relaxed hemoproteins (according

to the terminology of Perutz et al. [36]) does not enhance the affinity of their heme group for CO. When cooled at - 60 "C, compound (19) in toluene or dichloromethane reversibly binds oxygen without iron oxidation, as recently reported for other models and single hemes in organic solutions at low [12,37 401 or room [29] temperature and for picket-fence ferroporphyrins at room temperature [14]. Similar studies of Chang and Traylor [32] show that their imidazole-pyrroheme binds 0 2 whereas their pyridinemesoheme does not. The formation of the oxygen complex at -60 "C involves the splitting of the dimer of compound (19). Although no quantitative data are available upon the competition between O2 and imidazole, this ability to bind 0 2 can be related to the synergic effect discussed by RougCe and Brault [21] in the case of the formation of CO deuteroheme, by Chang and Traylor [32] and Geibel et al. [29], which stabilizes the oxygen complex with respect to the dimer. CONCLUSION The physicochemical properties we have reported show the important role of the electronic structure of the base upon the reactivity of five-coordinate hemoprotein models towards externally added ligands. The monomer-dimer equilibrium of the neutral oxidized form reflects that the six-coordinate ferric porphyrins are more stable than the five-coordinate species. This is corroborated by the low affinity constant of ferric porphyrins for the fifth ligand. Thus the histidine methyl ester attached to the side-chain is not firmly bound to the iron ion. However, the similarity between the absorption and ESR spectra of the ferric model derivatives and those of the same myoglobin derivatives unambiguously shows that histidine methyl ester becomes coordinated in these unsymmetrical compounds. The presence of histidine methyl ester firmly bound to iron in the reduced form shows the good stability of five-coordinate ferrous complexes, precluding the dimerization equilibrium at room temperature except at high concentration. The trans effect exerted by histidine methyl ester is not restricted by the side-chain as shown by the fact that the affinity of Fe"Dtp(OMe,His-OMe) (compound 19) for imidazole and CO is not decreased as compared with mono-(imidazo1e)deuteroheme dimethyl ester (compound 29). Like the latter it appears more representative of the reactivity of five-coordinate, unconstrained hemoproteins than other less reactive models described up to now.

REFERENCES 1. Perutz, M. F. (1970) Nature (Lond.) 228, 726-7734, 2. Dickerson, M. F. (1972) Annu. Rev. Biochem. 41, 815-842

16

M. Momenteau, M. Rougee, and B. Loock: Five-Coordinate Iron-Porphyrin

Perutz, M. F. (1969) Proc. R. Soc. (Lond.) B173, 113- 140. Kendrew, J. C. (1963) Science (Wash. D.C.) 139, 1259- 1266. Watson, H. C. (1969) Prog. Stereochem. 4, 299-307. Perutz, M. F. & Mathews, F. S. (1966) J . Mol. Biol. 21, 199202. 7. Wang, J. H. (1958) J . Am. Chem. Soc. 80, 3168-3169. 8. Lautsch, W. (1958) Kolloid Z. 161, 1 - 12. 9. Sano, S., Ikeda, K. & Sakakibara, S . (1964) Biochem. Biophys. Res. Commun. 15,284- 289. 10. Momenteau, M. & Loock, B. (1974) Biochim. Biophys. Acta, 343, 535- 545. 11. Warme, K. & Hager, H. P. (1970) Biochemistry, 9, 1599-1605. 12. Chang, C. K. & Traylor, T. G. (1973) Proc. Natl Acad. Sci. U.S.A. 70,2647-2650. 13. Castro, C. E. (1974) Bioinorg. Chem. 4, 45-65. 14. Collman, J. P., Gagne, R. R., Halbert, T. R., Marchon, J. C. & Redd, C. A. (1973) J . Am. Chem. Soc. 95, 7868-7870. 15. Caughey, W. S., Alben, J. O., Fujimoto, W. Y. & York, J. L. (1966) J . Org. Chem. 31, 2631 -2640. 16. Momenteau, M. (1973) Biochim. Biophys. Acta, 304, 814-827. 17. Brault, D. & Rougee, M. (1974) Biochemistry, 13, 4591 -4597. 18. Szwarc, M., Levy, M. & Milkovitch, R. (1956) J . Am. Chem. SOC.78,2656 - 2657. 19. Lexa, D., Momenteau, M. & Mispelter, J. (1974) Biochim. Biophys. Acta, 338, 151 - 163. 20. Maxwell, J. C. &Caughey, W. S. (1976) Biochemistry, 15, 388396. 21. Rougee, M. & Brault, D. (1975) Biochemistry, 14, 4100-4106. 22. Brault, D. (1976) These de Doctorat d’Etat, Paris. 23. West, W. & Pearce, S. (1965) J . Phys. Chem. 69, 1894- 1903. 24. Rossi-Fanelli, A. & Antonini, E. (1957) Arch. Biochem. Biophys. 72,243 - 249. 3. 4. 5. 6.

25. Brault, D. & Rougee, M. (1974) Biochim. Biophys. Res. Commun. 57,654- 659. 26. Tamura, M., Asakura, T. & Yonetani, T. (1973) Biochim. Biophys. Acta, 295, 467-479. 27. Momenteau, M., Mispelter, J. & Lexa, D. (1973) Biochim. Biophys. Acta, 320, 652-662. 28. Peisach, J., Blumberg, W. E., Wittenberg, B. A. & Wittenberg, J. B. (1968) J . Biol. Chem. 243, 1871-1880. 29. Geibel, J., Chang, C. K. & Traylor, T. G. (1975) J . Am. Chem. SOC.97, 5924- 5926. 30. Peisach, J., Blumberg, W. E., Ogawa, S., Rachmilewitz, E. A. & Oltzik, R. (1971) J . Biol. Chem. 246, 3342-3355. 31. Chang, C. K. & Traylor, T. G. (1973) J . Am. Chem. SOC. 95, 8475 - 8477. 32. Chang, C. K. & Traylor, T. G. (1973) J . Am. Chem. SOC.95, 8477 - 8479. 33. Antonini, E. & Brunori, M. (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands, Amsterdam, North-Holland. 34. Brunori, M., Noble, R. W., Antonini, E. & Wyman, J. (1966) J . Biol. Chem. 241, 5238-5243. 35. Amiconi, E., Antonini, E., Brunori, M., Fomaneck, H. & Huber, R. (1972) Eur. J . Biochem. 31, 52-58. 36. Perutz, M. F., Ladner, J. E., Simon, S. R. & Ho, C. (1974) Biochemistry, 13, 2163-2173. 37. Wagner, G. C. & Kassner, R. J. (1974) J . Am. Chem. SOC.96, 5593-5595. 38. Brinigar, W. S. & Chang, C. K. (1974) J . Am. Chem. Soc. 96, 5595- 5597. 39. Brinigar, W. S., Chang, C. K., Geibel, J. & Traylor, T. G. (1974) J . Am. Chem. Soc. 96, 5597-5599. 40. Almog, J., Baldwin, J. E., Dyer, R. L., Huff, J. & Wilkerson, C. J. (1974) J . Am. Chem. SOC.96, 5600-5601.

M. Momenteau and B. Loock, Section de Biologie, Fondation Curie, Institut du Radium, 15 Rue Georges-Clemenceau,F-91405 Orsay, France M. Rougee, Laboratoire de Biophysique du Museum National d’Histoire Naturelle. 61 Rue Buffon, F-75005 Paris, France

Five-coordinate iron-porphyrin as a model for the active site of hemoproteins. Characterization and coordination properties.

Preparation of iron(III)-deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been...
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