Models of the cytochromes-b and related heme proteins

Pror Indian Acad. Sei. (Chem. Sci.), Vol. 102, No. 3, June 1990, pp. 353-364.

9 Printed in India.

Models of the cytochromes-b and related heme proteins

O K M E D H I

Department of Chemistry, Gauhati University, Guwahati 781 014, India.

Abstract. Cytochromes-b, which are a class of heine proteins that have bis(imidazole) coordination to the so-called b-type heme(protoheme), show a wide variety of physical properties with apparently little change in the iron coordination environment. For example, the EPR of cytochrome-bs from liver (gm.~ = 2.9) is different from that of mitochondrial bs~o(gm,, = 3"78) though both proteins have a similar iron coordination environment.

We have studied Fem (PPIX) adducts with imidazole and substituted imidazoles in various solvents. Combined application of techniques such as Mfssbauer, EPR, and electronic spectroscopy show that the iron electronic structure in the bis(imidazole) models is influenced by steric factors of imidazole coordination and by the relative orientation of the axial imidazole planes. A change in the Mrssbauer quadrupole-splitting value from 2.43 rams- 1 to 1-87 rams- ~ and EPR gmax from 2"7 to 3"8 may be associated with the change of orientation of the imidazole plane from parallel to perpendicular.

Mrssbauer studies on the bis(histidine) complex of hemin show that histidine binds as a sterically hindered imidazole and that the iron-imidazole bonds are weak. The quadrupole- splitting value of 2"15 rams-i suggests non-parallel orientation of the imidazole planes in the model complex. Mrssbauer spectra and quadrupole splitting results are similar to those found in the low-spin ferric cytochromes and cytochrome-b5.

Keywords. Cytochromes-b; heine proteins; his (imidazole) coordination; imidazole planes;

quadrupole-splining.

1. Introduction

K n o w l e d g e of v a r i o u s factors t h a t influence the electronic structure of iron in h e m e proteins is of considerable significance in u n d e r s t a n d i n g the s t r u c t u r e - - f u n c t i o n relationship of metalloproteins. T h e n a t u r e of the axial ligands b o u n d to the hemes a n d the substituents o n the pyrrole positions h a v e been k n o w n to affect redox potentials, electronic a n d m a g n e t i c r e s o n a n c e spectra o f various types of heroes in proteins (Falk 1964; Smith 1975; D o l p h i n 1979; L e v e r a n d G r a y 1983). H o w e v e r , the c y t o c h r o m e s - b are a n interesting class of proteins where, even with a c o m m o n set of axial ligands a n d h e m e substituents, the physical p r o p e r t i e s of h e m e proteins v a r y widely. F o r e x a m p l e c y t o c h r o m e - b 5 f r o m liver ( M a t h e w s et al 1979) a n d e r y t h r o c y t e s (Passon et al 1972; I y a n a g i 1977) o f a n i m a l s a n d yeast f l a v o c y t o c h r o m e - b 2 (Labeyrie et al 1966; Keller et ai 1973) have very similar N M R spectra a n d well-characterized E P R spectra (Watari et al 1967; B o i s - P o l t o r a t s k y a n d E h r e n b e r g 1967; B l u m b e r g a n d Peisach 1971) typical of "class b" h e m e proteins with 0~ = 2.9, g2 = 2.2, a n d g3 = 1.5.

H o w e v e r , within the s a m e c y t o c h r o m e s - b class there are a wide variety of m e m b r a n e - b o u n d proteins, including m i t o c h o n d r i a l bs66(br) a n d bs62(br), c h l o r o p l a s t b6 a n d chloroplast bss9 which have E P R signals at gm,x > 3"3 as the sole o b s e r v a b l e spectral feature (Labeyrie et al 1966; B o i s - P o l t o r a t s k y a n d E h r e n b e r g 1967; W a t a r i et al 1967;

353

CH2 CH3 H CH--'~ CH2

H ! ~ CH3

CH3 ~ @ ~ @ CH3

CH 2 H C~

/

figure 1. Structure of (protoporphyrinato-lX) iron(III), heme b.

Blumberg and Peisach 1971; Passon et al 1972; Keller et al 1973; Iyanagi 1977;

Dolphin 1979; Mathews et a11979; Tsai and Palmer 1982, 1983; Lever and Gray 1983;

Salerno 1983, 1984).

It is well-known that all the proteins within the cytochromes-b class have bis(imidazole) coordination (Labeyrie et al 1966; Passon et al 1972; Keller et al 1973;

lyanagi 1977; Moore and Williams 1977; Mathews et al 1979; Widger et a! 1984;

Babcock et al 1985) to the so-called b-type heme (figure 1). The wide range of physical properties of cytochromes-b with apparently little change in the iron coordination environment is believed to be due to three reasons. First, it is speculated that a change in the relative orientation of the planes of the two imidazole ligands that are bound to the iron porphyrin moiety may account for the differences in the EPR spectra (Tsai and Palmer 1982; Walker et al 1984, 1986; Widger et a! 1984; Scheidt and Chipman 1986). Second, "strains" in bis(histidine) ligation may be responsible for the unusual so-called highly anisotropic low-spin (HALS) EPR signals with gmax > 3"3 (Carter et al 1981; Palmer 1983). Third, hydrogen bonding to the N - H of the coordinated imidazole (of histidine) may be responsible for modulation of the iron electronic structure within the same coordination environment (Brautigan et al 1977;

Quinn et al 1984; O'Brien and Sweigert 1985).

Though the b/s(imidazole) complexes of iron(Ill) porphyrins have been widely studied previously by various techniques (Dolphin 1979; Lever and Gray 1983) there have been relatively few M6ssbauer spectroscopic studies on the protoporphyrin-lX analogues (Epstein et al 1967; Bullard et al 1974; Sams and Tsin 1979). Moreover, the accuracy of some of the earlier M6ssbauer data (Bullard et al 1974) and their interpretation have been questioned (Sams and Tsin 1979). In the light of the recent interest in cytochromes-b, we have carried out a systematic M6ssbauer spectroscopic study on the bis(imidazole) complexes of (protoporphyrinato-IX) iron(Ill) because this experimental technique provides unambiguous characterisation of the iron electronic structure (Sams and Tsin 1979). The aim of the studies described here was to gather evidence to test the various hypotheses of steric and/or electronic influence

Cytochromes-b and related heme proteins 355 of the imidazole ligand on the heme electronic structure. An interesting correlation between EPR and M6ssbauer spectral data is found for the iron(III) complexes reported here.

There is no doubt that the primary control of heine iron reactivity in hemeproteins involves the steric and/or electronic influence of the ubiquitous histidyl imidazole ligand (Perutz and Ten Eyck 1971), which is a common axial ligand for all hemoproteins. In this study we also report the effect of various hindered and non-hindered imidazoles, including histidine, on the iron(Ill) electronic structure of (protoporphyrinato-IX) iron(Ill)l(PPIX)Fe(IlI)] complexes in solution. Histidine seems to behave like a sterically hindered imidazole in these models.

2. Experimental

Since it is well-known that iron(IIl) protoporphyrin-IX (hemin) undergoes extensive aggregation in water, the choice of the solvent and reaction conditions were worked out so as to obtain maximum solubility and stability of the monomeric bis(imidazole) complexes. In almost all the cases a bemin concentration dependence of the visible absorption band near 410rim gave clear indication of the presence of monomeric bis(imidazole) complexes in solvents such as dimethylsulphoxide (DMSO) and 1:1 ethanol-water mixture. A thousandfold excess of ligands (figure 2), imidazole(ImH), 1-methylimidazole (1-MeIm), 2-methyl-imidazole(2-MeImH), or histidine(His), was used in order to drive the ligand-binding equilibria towards the exclusive formation of the low-spin bis(imidazole) species in solution. The electronic spectra compare favourably with those reported previously for similar complexes (Pasternack et al

1978).

ZmH RI:H 9 R2:H P R3:H

1MeIm R,I:CH 3 ~ Ag: H : R3= H

2MeImH Histidine

(His) Figure 2.

RI:H 9 R2=CH3 9 R3=H H

%-

i i

NH 2

Structure of imidazole derivatives relevant to this work.

Frozen solutions of M6ssbauer samples were prepared by using 57Fe enriched heroin (Caughey et a11966) and the spectra recorded using instruments and techniques previously described by us (Medhi et al 1989).

3. Results and discussion

3.1 Quadrupole splittinffs in low spin ferric porphyrins

In the following we briefly consider the important features of the electronic structure of low-spin iron(III) porphyrin complexes so as to put our experimental results into proper perspective.

In the bis(imidazole) complexes of iron(III) porphyrins the degeneracy of the one-electron states of low-spin d 5 (t~o) ion is lifted by axial and rhombic perturbations to give dxy as the most stable followed by dxz and dy= in order of increasing energy (Palmer 1983) (figure 3). The hypedine parameters such as magnetic hyperfine field and electric field gradient (EFG) at the nucleus may be calculated by using such a model (Golding 1969; Oosterhuis and Land 1969). Golding (1969) showed that for maximum rhombic distortion the value of AEQ is 2"5 mms-1 for low-spin iron(III) compounds.

In order to relate E F G to molecular structure and bonding the valence contribution to EFG (qva~) may be expressed as

qval = qcF + qMO,

where qcv, the crystal field term, results from the population of one-electron st/~tes in figure 3, and qMo originates from the donation/withdrawal of charge density due to covalency. The relative changes in qv,~ in various compounds may then be estimated qualitatively by using the Townes-Dailey approximation (Dailey and Townes 1955) and chemical intutition of o-donation and/or n-bonding by the ligands. Since the qcv term dominates AEQ values in low-spin iron(Ill) systems (Johnson and Shepherd 1983), we suggest that in the bis(imidazole) complexes of heroin the symmetry of the charge distribution in the d-orbitals (hence qcv) is mainly controlled by the

A, lu W Z

~.ey z

TII

,

RHOMBIC ( 0 4 ) AXIAL ( r

Q b

Figure 3. One-electron energy states (d- orbitals) and the ligand field parameters for low-spin iron(Ill) porphyrins in rhombic (a) and axial (b) symmetry perturbations. VIA and A/~. are the rhombic and the axial symmetry parameters, respectively, expressed in the units of spin-orbit coupling constant (~).

Cytochromes-b and related heme proteins 357 magnitute of the rhombic distortion parameter (V) in relation to the spin-orbit coupling constant (3.).

In order to relate M6ssbauer quadrupole-splitting with the EPR g-values, two situations may be considered.

(A) When V >> 3. and A >> 3., the ground state is an orbital singlet with the unpaired electron localized in the dyz orbital (figure 3a). The unequal distribution of charge density in the x and y directions give rise to a large qcF and, in the absence of any contribution from quo and q~uice, this situation corresponds to Golding's (1969) value of maximum AEQ of 2.5 mms-~ for maximum distortion in a low-spin iron(III) complex. The EPR spectrum of such a compound would show a three-line feature typical of "class b" hemoproteins (Migita and Iwaizumi 1981; Palmer 1983). Such a situation arises when the two imidazole planes are in a parallel orientation (figure 4a) and both the imidazole ligands interact with the iron dy~ orbital so as to make this the highest energy orbital (figure 3a).

(B) When V < 3, and A >> 3., the unpaired electron is delocalized over the dx~ and dy, orbitals giving rise to an orbital doublet ground state for the complex. The dx~ and dyz orbitals attain near degeneracy (figure 3b) and the effective electronic symmetry (hence qcr and AEQ) is smaller than that described in (A) above. For maximum n-overlap, the axial imidazole ligands should be oriented perpendicular to each other (figure 4b) so that one of the imidazole ligands forms a n-bond with the iron dyz orbital and the other forms a n-bond with the iron d~, orbital. Crystal structures (Scheidt et al 1967; lnniss et al 1988) of [(TPP)Fe(2-MelmH)2] § and [(TPP)Fe(Py)2] + show that the two planar axial ligands adopt a perpendicular geometry and analyses of EPR data for similar compounds (Migita and Iwaizumi 1981; Palmer 1983; Walker et al 1986; lnniss et al 1988) show near degeneracy of the d~, and d~ orbitals. The EPR spectra are typical of HALS systems with #rex > 3.3 (Palmer 1983; Migita and lwaizumi 1981). The reported M~Sssbauer data for such compounds (Walker et al 1984; Sams and Tsin 1979) show that the AEQ value is about l'9mms -1 for the perpendicular orientation of axial ligand planes.

3.2 Effect of imidazole plane orientation on AEQ in [ ( P P I X ) F e ( I m R ) 2 ] +

The quadrupole-splitting of the 2-MeImH complex in table 1 is the lowest (AEQ = 1.87mms -1) in the series of compounds reported here. These results compare favourably with those of [(PPIX) Fe(Py)2 ] + (EQ = 1.88 mms - 1 ) (Carter et al 198 l) and [(TPP) Fe(2-MelmH) 2 ] + (AEQ = 1-77 rams- 1) (Walker et al 1986) both of which also show the HALS EPR spectrum (Walker et al 1986; Migita and Iwaizumi 1981) with

#max > 3"0. As discussed earlier [c.f. (B) above] these results are consistent with a near degeneracy of dy~ and dxz orbitals and a molecular geometry where the planar axial ligands are in perpendicular orientation. The crystal structure of the 2-MeImH complex shows a near perpendicular orientation of the imidazole planes (Scheidt et al

1987).

The AEQ value of[(PPIX)Fe(III)(l-MeIm)2] + is highly solvent dependent (table 1).

In frozen aqueous ethanolic solution this compound manifests a sharp symmetric quadrupole-doublet (AEQ = 2.34 mms- ~) in contrast to a broad asymmetric doublet (AEe = 2.24 mms-1) in DMSO. The EPR data show that in 1:1 ethanol-water the rhombic distortion and rhombicity are much larger than in DMSO solution.

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Cytochromes-b and related heme proteins 359

Table I. M6ssbauer, EPR data and crystal field parameters for [(PPIX)Fe(IIIXImRh] + complexes a.

Compound

6

(Fe) AE e

Solvent (mms- 1) (mms- 1) 0m., VIA A/~

[(PPIX) Fe(III)(Im)(ImH)] ~ [(PPIX) Fe(III)(ImH)2] + [(PPIX)Fe(III)(I-Melm)2 ] + O H - b [(PPIX) Fe(llI)(1-Melm)2 ] + [(PPIX) Ee(III)(2-Melm)2] + [(PPIX)Fe(III)(His)2 ] + Cytochrome-b 5, liver, native r

DMSO 0-24(3) 2.43(3) 2"76 2 - 4 5 3"71 DMSO (}22(2) 2"38(2) 3"02 1 " 7 7 3-54 1:1 Ethanol: 0.26(1) 2"34(1) 2-74 2 " 5 7 2"80

water

DMSO 0.23(1) 2-24(1) 2"97 1.84 3"27 1:1 Ethanol: 0.16(2) 1"87(2) 3.48 1 " 1 5 2"26

water

1:5 Ethanol: 0.24(2) 2"15(2) 2"92 1'39 2.86 water

Alkaline ~ d 2.76 2 " 4 5 3"71 (aqueous)

Neutral 0-23(3) 2"27(3) 3"03 1 . 6 8 3"23 a M6ssbauer data at 80 K, EPR data at 4 K, and crystal field parameters are defined in figure 3 and are calculated from the EPR g-values using Taylor's method (Palmer 1983); bFormulation from Little et al (1973) (see text); ~ M6ssbauer data at 195 K 48 and EPR data at 4 K (Peisach and Mims 1977); d Not known.

The crystal structure of [(PPIX) Fe(1-MeIm)] § OH" H 2 0 shows that the two axial ligands are nearly parallel to each other (Little 1975). Both C H 3 O H and H 2 0 molecules are hydrogen-bonded to the propionate carboxylate groups of PPIX. Based on this, and the preceeding discussion [c.f. (A) and (B) above], we suggest that the 1-Melm complexes have different imidazole-plane orientations in the two solvents studied. In 1:1 ethanol-water, the C 2 H s O H and H 2 0 molecules form hydrogen bonds with the propionate groups and stabilize the near-parallel orientation as in the crystal structure. This situation is reflected in a larger value of AEQ, smaller value of gma, and a larger rhombic distortion of the complex in 1:1 ethanol-water solution.

In frozen DMSO solution, the values of AEQ, 0rex and rhombic distortion are, however, smaller than expected for perpendicular orientation as in the 2-MeImH analogue (table 1). Hence the orientation of 1-Melm planes in D M S O is non-parallel though they do not approach perpendicular orientation. The change in imidazole-plane orientation in the two solvents may be related to the strength of the Fe-imidazole bonds which are weaker in DMSO (stability constant for imidazole binding,//2 "~ 104 M-2, as compared to that in 1:1 ethanol-water mixture, f 1 2 m 1 0 6 M - 2 ) . A weaker bond in DMSO allows some degree of freedom for rotation along the Fe-N(imidazole) bond axis. Thus the bis(1-Melm) complex in DMSO may be compared to the 2-MelmH analogue where the steric hindrance of the axial ligand binding to the heine leads to greater Fe-N(ImH) distance and non-parallel orientation of the imidazole planes. Thus steric factors leading to a greater Fe-N(imidazole) distance may be responsible for non-parallel orientation as evidenced by small AEQ and large gmax values [AEQ approaching 1"9 mms-1 and gmax approaching 3"5 in the bis(2-MeImH) analogue].

The quadrupole splitting of [(PPIX)Fe(ImH)2] + is independent of the nature of solvents and show a value of 2-38 + 0.02 mms- t. This value is similar to that of the 1-Melm analogue in aqueous ethanol as described above and a similar imidazole-plane

orientation may by predicted. On deprotonation of one of the imidazoles a large value of AEQ(2.43 mms - t ) was obtained which probably corresponds to a parallel orientation of imidazole planes. From the M6ssbauer data we expect a structure with large rhombic distortion, which was indeed observed from the analysis of the EPR spectrum of this compound (table I). It may be pointed out that in the crystal structure of [(TPP)Fe(ImH)2 ] + the angle between the imidazole planes was found to be very large (Collins et al 1972) (0 = 57~ which is different from that inferred from the M6ssbauer and EPR data of the PPIX analogue. This difference may be due to the fact that in frozen solution the imidazole planes tend to attain the most thermodynami- cally stable parallel orientation, whereas in the crystal structure of the TPP analogue a large angle between the planes of ImH may be due to the influence of crystal-packing forces.

Effects of the hydrogen bonding of the axial imidazole ligands on M6ssbauer parameters have been discussed by us recently (Medhi and Silver 1990). We found that such hydrogen bonding favours non-parallel orientation of the imidazole planes, influences the basicity of the axial ligands, and stabilizes the low-spin states of the ferric ion (Medhi and Silver 1990).

3.3 Bis(histidine) complexes of hemin

Dependence on pH of the M6ssbauer spectral data of the bis(histidine) complex of (protoporphyrinato IX) iron(III) in frozen aqueous ethanolic solution, in the range 7"5-10"1, shows that at the millimolar concentrations used for the M6ssbauer study, the low-spin iron(Ill) complex [(PPIX)Fe(III)(His)2] + is the major species at pH 8"0.

In the pH range 8"0 < pH < 7.5 one of the histidine ligands is displaced by an O H - ion or H 2 0 molecule and aggregated species are formed.

The isomer shift (0"24mms - l ) and quadrupole-splitting (2.15rams - t ) of the bis(histidine) complex (table l) and the broad asymmetric line of the quadrupole doublet (figure 5) are similar to those of the bis adducts of hindered imidazoles such as 2-MelmH or l-MeIm in DMSO. The spectral line shape and the AEQ value of the bis(histidine) complex is clearly different from that of the analogous unsubstituted imidazole derivatives (table I).

Comparing the AEQ values and the spectral live shapes of the bis(histidine) complex with those of the substituted imidazole analogues, we found that the histidine binds as a sterically hindered imidazole ligand, such as 2-MeImH. Though the side chains of histidine should not impose as severe a steric restriction as the methyl groups in 2-MelmH, the electrostatic and hydrogen-bonding interactions of the amino acid side-chain of histidine with the propionic acid group of PPIX may lead to some steric strain in histidine coordination to hemin. The steric strain in bis(histidine) ligation and the weak Fe-N(imidazole) bond is responsible for the AEQ value of 2.15 mms-1 which indicates a non-parallel orientation of the imidazole-planes with a large angle between them.

The two lines of the quadrupole-doublet in the M6ssbauer spectrum (figure 5) of the bis(histidine) complex are of unequal intensity. Though the areas of both the lines are equal the higher velocity line is unusually broad (FWHM are 0.48(2) and 0"66(3)mms - t for the low and high velocity lines respectively). Such spectra are usually obtained for heme proteins and the cause of the asymmetric line broadening is attributed to slow spin-lattice relaxation rates (compared to the nuclear precession

Cytochromes-b and related heme proteins 361

I I

o ,~ o.

0 ( h ( h

0 Ch ( h

e,-

.

::.

9 e

e "b t 90 ,.~

4 ~ e

4 ~ o o 9

. . . ~ .

4 e 4 p~ 9

9 4 q q i I 9 q~

eq~

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~84e e e ~ 4 e , e

9 ~t t 9

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frequency) (Bradford and Marshall 1966; Lang et al 1966; Lang and Marshall 1966;

Medhi and Silver 1990).

3.4 Relevance to cytochromes-b

The Mrssbauer parameters of the model compounds described here agree quite well with those reported (Miinck 1978) for cytochrome-bs. Miinck has reported that the Mrssbauer spectrum of cytochrome-bs at low temperature is broad (Miinck 1978) and that the slow relaxation in b5 is similar to that (Lang et al 1966) in cytochrome-c.

Such broad and asymmetric lines are observed by us in the bis(l-Melm) complex in DMSO, in bis(2-MelmH) and in his(His) adducts of hemin in aqueous ethanol. The steric strain imposed by the side-chains of the histidine ligands in cytochrome-b5 may be responsible for the observed Mrssbauer spectrum of the protein. Comparison of the quadrupole-splitting (AEQ = 1.9 - 2.2 mms- 1) of the hindered imidazole complexes (including histidine) with that in the protein (AEQ = 2.27 mms- 1) suggest a non-parallel orientation with a large angle between the axial ligand planes.

The EPR spectrum of cytochrome-b5 show that the protein exists in two different forms in neutral (gz = 3-03, gy = 2.23, gx = 1.43) and alkaline (gz = 2.76, #y = 2.28, #x = 1.68) media (Peisach and Mims 1977). The EPR spectrum of the complexes with hindered imidazoles and histidine are comparable to those ofcytochrome-bs in neutral solution, while for the non-hindered imidazole adducts the EPR spectra are typical of those ofcytochrome-b5 in alkaline media. The results for the bis(1-Melm) complex in table 1 show a transition between these two situations in aqueous ethanol versus DMSO.

Based on the Mrssbauer and EPR spectral studies of the model complexes described here, we attribute these two situations to be due to two different orientations of the axial imidazole planes (figure 4). In neutral solution of the protein and in the model complexes of sterically hindered imidazoles, including histidine, the two imidazole planes are in non-parallel orientation with a large angle between the imidazole planes, whereas in alkaline solutions of the protein and in the model complexes with sterically hindered imidazoles the imidazole planes are in near-parallel orientation.

The results for the histidine and 2-MelmH complexes indicate that the non-parallel orientation of the two imidazole planes arise due to steric reasons as well as a weak Fe-N(imidazole) bond. In the model bis(histidine) complexes studied here, such steric forces could not be eliminated to obtain the parallel orientation of imidazole planes, whereas alkaline solutions of the protein have a near parallel orientation of axial ligand planes. The question to be asked is: what makes the parallel orientation of imidazole planes possible in cytochrome-b5 and in chloroplast cytochrome-bssg?

Though hydrogen bonding and deprotonation of N - H of histidine is a possibility (Medhi and Silver 1990), this is not always necessary since a change in the imidazole-plane orientation was also found in the 1-Melm complex where the axial ligands can not form hydrogen bonds. Interestingly, neither l-Melm nor histidine is as stericaUy hindered (Lever and Gray 1983) as 2-MelmH in coordinating to the heme, the steric effect of histidine coordination is due to the interaction of the amino acid side-chain with the heme. In a protein such interactions may be modulated by the apoprotein conformation so as to stabilize the parallel (or perpendicular) orientation of the imidazole planes.

Cytochromes-b and related heme proteins 363 4. Conclusion

The orientation of planar axial ligands and various steric and/or electronic factors have considerable influence on the iron electronic structure of ferrihcmes. A value of AEQ = 2.43 mms- 1 is assigned to a parallel orientation of the planar imidazole ligands, while a much lower value of AEQ = 1.87 mms- 1 is assigned to a situation where the planar ligands are in a perpendicular orientation. In order to stabilize the perpendicular orientation of the ligand planes considerable steric interaction between the porphyrin and the axial ligands is necessary.

Histidine binds to heme as a sterically hindered imidazole, where the steric hindrance may be due to the electrostatic and hydrogen-bonding interactions of the amino acid side-chain with the propionic acid carboxylate groups of PPIX. This, together with a relatively longer iron-imidazole bond may be responsible for aligning the two imidazole planes in a non-parallel orientation with a large angle between the planes in the bis(histidJne) complex (AE e = 2.15 mms- 1). Thus, the steric strain in histidine coordination, solvent effects, and the influence of charged groups near the beme in cytochromes-b and related heine proteins may play a combined role in orienting the planes of the two histidyl imidazole ligands of the heme prosthetic group.

Acknowledgements

We thank the Association of Commonwealth Universities, London, for financial support and Dr Jack Silver of Essex University, UK, for providing the facilities for M6ssbauer work.

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