Electron paramagnetic resonance studies of highly anisotropic low-spin states of ferrimyoglobin derivatives

290

Biochimiea etBiophysieaActa. 743 (1983) 290 298

Elsevier Biomedical Press BBA 31551

E L E C T R O N P A R A M A G N E T I C R E S O N A N C E S T U D I E S OF H I G H L Y A N I S O T R O P I C L O W - S P I N

STATES OF FERRIMYOGLOBIN DERIVATIVES CATHARINAT. MIGITA a.,, KOUTO MIGITA b and MASAMOTOIWAIZUMI'~ '~ The Chemical Research Institute of Non -Aqueous Solutions, Tohoku University, Sendai 980 and h School of Education, Yamaguchi University, Yamaguchi 753 (Japan)

(Received October 13th, 1982)

Key words." ESR," Myoglobin," Denaturation," Highly anisotropic low-spin state," Nitrogenous base

The effects of addition of nitrogenous bases, which gave low-spin ferric porphyrin complexes with highly anisotropic g values, were investigated for ferrimyoglobin by low-temperature EPR measurements. Concomitant denaturation of myoglobin upon addition of the exogenous bases was also of interest. By addition of pyridine-type bases under regulated pH, Mb(Fe 3 +) complexes showing EPR spectra with highly anisotropic g values were formed. These complexes have the electronic states close to the spin-crossover point but not so close as that of the ferric porphyrin highly anisotropic low-spin (HALS) complexes previously reported. Several types of low-spin species, LSi, LS a and LSb, were produced by the denaturation of myoglobin caused by addition of some exogenous ligands. The LS i was assigned to a complex with histidine-E7 coordinated on the sixth position and LSa to the one with O H - and histidine-F8[lm°l .

Introduction The investigation of electronic states of haem systems and their interconversion, both in model complexes and in haemoproteins, is of great interest for obtaining a detailed understanding of their biological function. From this point of view, detection and confirmation of haem complexes in the intermediate states both electronically and structurally are fundamentally important. In a recent paper [1], we demonstrated by lowtemperature EPR studies that ferric protoporphyrin IX complexes with a series of nitrogen heterocyclic compounds as axial ligands provide

* Present address: Department of Chemical Engineering, Faculty of Engineering, Yamaguchi University, Ube 755, Japan. Abbreviations: HALS,highlyanisotropic low-spin species; Mb, myoglobin; LS, low-spin species; L¢x, exogenous ligand; HS, high-spin species. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

highly anisotropic EPR spectra which differ from well-known low-spin spectra. Analysis of the g values of these complexes led to the conclusion that their electronic states are very close to the spin-crossover point. The reverse correlation between the axial field strength and the tetragonal splitting among the complexes and the temperature dependence of the spectra suggested that the structure around the haem iron in these specific low-spin complexes must be similar to that of high-spin complexes. This specific low-spin state, denoted HALS for convenience, was realized in ferric porphyrin systems either when two moderate axial ligands were coordinated or when one strong ligand and one weak ligand were coordinated. That is, a moderate cubic field, weaker than in the typical low-spin state and stronger than in the high-spin state, is indispensable for the HALS state. This 'intermediate' state seems to occur also in haemoproteins. Brauti_gan et al. [2] and others

291

[3-5] have observed EPR signals with high g values similar to the low-field part of the HALS spectra for cytochromes. However, the analysis of the EPR signals did not lead to a full understanding of the nature of the EPR signals of high g anisotropy, and the relationship between the g anisotropy and the tetragonal splitting evaluated by Brautigan et al. [1] is not consistent with our estimation of the relationship in HALS complexes. Such inconsistency with our result has also been seen in Dickinson et al.'s report [6] for carp ferrihaemoglobin. In the present work, we intended firstly to substantiate the HALS state in ferrimyoglobin derivatives and to confirm our previous assertion [ 1] on the relationship between the axial field strength, the g anisotropy and the tetragonal splitting. Secondly, we wanted to clarify the contribution of the globin part to the structural and electronic characteristics of the haem-ligand moiety in fer-

rimyoglobin-exogenous ligand complexes by comparing the model-HALS systems with the MbHALS systems. Finally, general features of the denaturation of ferrimyoglobin by the exogenous ligands were examined. Materials and Methods

Materials. Equine skeletal muscle myoglobin was purchased from Sigma (type I) and used without further purification. Compounds used as exogenous ligands were methanol, pyridine, 2-, 3and 4-methylpyridine, 2-, 3- and 4-aminopyridine, 4-cyanopyridine, 1,4-diazine, pyrazole, imidazole, and 1-, 2- and 4-methylimidazole. They were all reagent grade and were purified by vacuum distillation for liquid compounds and by sublimation for solid compounds, whenever needed. Solvent water was a redistillate of distilled water commercially obtained.

TABLE I CLASSIFICATION OF THE PRODUCTS OBTAINED BY ADDITION OF EXOGENOUS LIGANDS TO FERRIMYOGLOBIN Py, pyridine (1 M) a; 4-MePy, 4-methylpyridine (0.2 M); 3-MePy (0.2 M); 3-NH2Py, 3-aminopyridine (0.4 M); 1,4-diazine (0.04 M); Pz, pyrazole (0.3 M); Im, imidazole (0.3 M); 4-Melm, 4-methylimidazole (0.3 M); 1-Melm (0.5 M); 2-Melm (0.2 M); 4-NH2PY (0.4 M); 2-MePy (0.06 M); 2-NH2Py (0.4 M); 4-CNPy; 4-cyanopyridine (0.06); MeOH, methanol (2 M); L~x, exogenous ligands; HS, high-spin species; LSi, low-spin species of Mb(Fe3+)(Im) type; LSa, low-spin species observed in acidic solution of Mb(Fe3+); LS b, low-spin species of Mb(Fe 3+)(OH-). Lex

Mb(Fe 3+)(L~) complex

Py 4-MePy 3-MePy 3-NH2PY 1,4-Diazine Pz Im 4-Melm l-Melm 2-MeIm 4-NH2Py 2-MePy 2-NH2Py 4-CNPy MeOH

main main main main sub main main main main

HS

LS i

LS b

LSa

sub sub

detected detected detected

sub minor main

sub main main main main main

(main) b (main) b (main) b minor sub sub sub

a The final concentration of each specific ligand used. [Mb] = 1 mM. b Mb(Fe3+)(Le,) = LSi.

Strongly denatured species

sub main

detected detected sub sub

292 The pH of sample solutions was adjusted by Horiba pH Standard Solutions which use phthalate for acidic, phosphate for neutral, and borate for basic pH. Sample solutions were prepared as follows: aqueous solutions of myoglobin and ligand compounds were prepared separatelY. The former was titrated with the latter until about 90% of the critical amount (as previously established) of the ligand compound needed to start denaturation of ferrimyoglobin was added. The pH of the resultant myoglobin-exogenous ligand solutions was measured by a Hitachi-Horiba M-5 pH meter. 0.3 ml of each solution was transferred to an EPR sample tube, made of high quality quartz and fitted with a glass joint for connection to a vacuum line in order to evacuate the sample solutions. No K 3Fe(CN)6 was added to the myoglobin solutions. The myoglobin concentration of each sample was about 1 mM and that of ligand bases was dozens to a few thousand times that of the myoglobin concentration, as indicated in Table I. Instrumentation. EPR spectra were measured with a Varian E-112 X-band spectrometer equipped with an Oxford ESR 9 cryostat. All the EPR spectra were recorded using 100 kHz field modulation and the microwave frequency was monitored by a Takedariken TR5501 frequency counter with a TR5023 frequency converter. The magnetic field was calibrated using an Okidenki WX-601C magnetofieldscope which was modified to monitor directly the proton resonance frequency with a Takedariken TR5578A universal counter. During EPR measurements, sample temperature was kept at 10 K and varied between 10 and 70 K whenever needed. Spectra simulation was made with a HewlettPackard 9825A calculator.

LSa A

5.2

B

7

C

8.1

HS

~ / x 9

J,-----4_

HS

GAIN 16

~xl

I

I

I

I

0.1

0.2

0.3

0.4

BIT Fig. l. pH dependence of EPR spectra of low-spin species obtained from ferrimyoglobin solutions. Each spectrum was recorded under the followinginstrumental setting: microwave frequency, 9.414+2 MHz; microwave power, 10 roW; field modulation frequency and amplitude, 100 kHz and 0.8 mT; time constant, 0.25 s; scanning rate, 25 mT/min; temperature, 10K.

convenience, we denote the low-spin species observed in the acidic and neutral solutions as LSa and that observed in basic solutions a s L S b. Here, LS b is the w e l l - k n o w n alkaline f o r m , Mb(Fe3+)(OH -) [7,8]. The origin of LSa will be discussed later.

Results

Product variation in the complex formation of ferrimyoglobin with exogenous ligands

pH dependence of EPR spectra of ferrimyoglobin solutions

The various products were detected by low-temperature EPR for the systems of ferrimyoglobin and exogenous ligands, as summarized in Table I. The products can be classified into six types. Some of the exogenous ligands, Lex, produced the Mb(Fe3+)(Le,) type of complexes. The weakly coordinating ligands seem to have only a minor effect on ferrimyoglobin, as presupposed, so that

Prior to examining effects of exogenous ligands, EPR spectra of ferrimyoglobin solutions of various pH were measured (Fig. 1). It is clear from these spectra that there exist two kinds of low-spin species in ferrimyoglobin solutions and they alternate depending on the pH of the solution. For

293 the main species in these systems was the high-spin ferrimyoglobin, HS, originally present. It is noteworthy that EPR spectra very similar to those of Mb(Fe3+)(imidazoles) were detected as by-products of the addition of non-imidazole bases such as pyridine derivatives to the ferrimyoglobin solutions (LS i shown in Fig. 2). On addition of strong bases such as 4-HH2-pyridine, alkaline form, Mb(Fe3+)(OH-), LSb, was mainly produced. Interestingly, low-spin species similar to that observed in acidic solutions, LSa, were also detected in Mb(Fea+)-4-CNpyridine and Mb(Fe3+)-MeOH systems. As a minor portion, EPR signals with g values about 3.4, which is characteristic of the HALS species in haemin systems, were observed in several systems (classified as strongly denatured species in Table I). These species with high g values may be attributed to the complexes formed between the bare haem resulting from strong denaturation and added ligands, because these species were very minor and the added ligands easily form

complexes with ferric protoporphyrin IX, having quite similar g values. Examples of the EPR spectra of LS,, LSb, LS i and Mb(Fe3+)(Lex) are shown in Fig. 2.

Characterization of Mb(Fe ~+)-HALS complexes by EPR features Among the Mb(Fe3+)(Lex) complexes listed in Table I, the complexes with pyridine, 4-Me-pyridine, 3-Me-pyridine, 3-NHz-pyridine and 1,4-diazine provided EPR spectra quite different from those of typical low-spin ferric complexes (Fig. 3).

gz

A

B

LSa H5

LSb

.ALSb ,

C.~..~ D~

V

Lex=Ira

V

D

~~~Lex=Pz I

I

i

0.3 0.4 0.5 BIT Fig. 2. EPR spectra obtained from ferrimyoglobin solutions containing: A, MeOH; B, 4-NH2-pyridine; C, imidazole; and D, pyrazole. Instrumental setting: microwavefrequency, 9.415 +3 MHz; microwave power, 10 mW; field modulation frequency and amplitude, 100 kHz and 0.8 roT; time constant, 0.25 s; scanning rate, 25 mT/min; temperature, 20 K. 0.1

0.2

I

0

I

I

0.2

i

I

0.4 BIT

,

I 0.6

,

I 0.8

Fig. 3. EPR spectra of Mb(Fe3+)-HALS complexescontaining: A, pyridine; B, 4-methylpyridine; C, 3-NH2-pyridine; and D, 1,4-diazine as exogenous ligands (Le~). Instrumental setting: microwave frequency, 9.408 MHz for trace A and 9.4154-2 MHz for traces B-D; microwavepower, 10 roW; field modulation frequency and amplitude, 100 kHz and 0.8 mT; time constant, 0.25 s; scanning rate, 50 roT/rain; temperature, 10 K.

294

The g anisotropy of these complexes is larger than those of common low-spin spectra of ferric porphyrin complexes such as A - D in Fig. 2. These spectra were undetectable above 30 K, whereas the common low-spin spectra are observable even at 77 K. These two characteristics are common to the HALS complexes of the ferric porphyrin system [1]. Therefore, we differentiate this type of Mb(Fe3+)(Lex) complexes from the others by describing it as Mb(Fe3+)-HALS.

(2)

~ = E(d~)- E(d~)

(4)

Here, the E ( d ) values are the respective d-orbital energies and they are expressed in units of the spin-orbit coupling parameter ~ of a ferric ion in haem. The parameters A and B are expressed in terms of the principal g values as follows:

Analysis of g values The three principal g values were assigned to g., g, and gx in order of decreasing g values, following the single-crystal studies of low-spin haem systems [9,10], as shown in Figs. 2 and 3. Using the g values obtained, the ligand field parameters of these low-spin ferrimyoglobin complexes were calculated according to Taylor's method [11]. The ligand field parameters, i.e., tetragonal splitting (A), rhombic splitting (V) and rhombicity (V/A), are defined as follows: Al= B - A / 2

V= A

A = g~,/(g~ + g v ) + g v / ( g ~ - g~)

(s)

B = g~/(g. + gy)+g:/(gy-

(6)

gx)

The ligand field parameters thus obtained for Mb(Fe3+)(Lex) complexes are listed in Tables II and III together with the principal g values obtained experimentally. Table II also contains our previous results for the ferric porphyrin complexes for comparison.

Spectral simulation for Mb(Fe 3 +)-HALS complexes Since HALS species have highly anisotropic g

(1)

TABLE I1 g VALUES A N D L I G A N D F I E L D P A R A M E T E R S OF F E R R I M Y O G L O B I N - E X O G E N O U S L1GAND COMPLEXES A N D F E R R I C P O R P H Y R I N COMPLEXES Ligand field parameters are defined in the text. Abbreviations as in Table I. V and Al are expressed in units of the spin-orbit coupling constant ?~ of a ferric ion in haem. Complex

Lex

gx

gy

gz

V

A

IV/All

Mb(Fe 3 + )-HALS

Py 3-N H 2 PY 4-MePy 3-MePy 1A-diazine Im Pz b Py 3-NH 2 PY 4-MePy 3-MePy 1,4-diazine Im Pz b

1.11 1.11 1.00 _ a 1.14 1.51 1.62 0.98 0.78 0.81 0.98 _ a 1.53 1.71

2.03 2.00 2.12 2.10 2.19 2.22 2.30 2.25 2.40 2.42 2.28 _ a 2.24 2.35

3.07 3.02 3.12 3.11 3.02 2.92 2.74 3.43 3.48 3.46 3.48 3.23 2.91 2.63

1.25 1.27 1.19

2.93 2.98 2.38

0.428 0.426 0.500

1.38 1.87 2.38 1.09 1.02 1.05 1.08

2.40 3.43 3.16 2.33 1.77 1.76 2.31

0.576 0.545 0.753 0.469 0.577 0.597 0.469

1.92 2.90

3.44 3.00

0.558 0.965

Mb(Fe 3 +)(Lex ) Fe3 +(PP)(L~×

)2 ~[HALS]

Fe 3 +(PP)(Le~ ) 2 [LS]

Unobservable. b The principal axes of the g tensor do not conform to the proper axis system presented in Ref. 11. See the text. ~ (Protoporphyrinato)iron(III) complex with Lex [1].

295 T A B L E III g VALUES, L I G A N D FIELD P A R A M E T E R S A N D L I G A N D A S S I G N M E N T OF LSi, LSa A N D LS b Abbreviations as in Table I. V and A are expressed in units of the spin-orbit coupling constant 2~ of a ferric ion in haem. Detected complex

Added Lex

gx

gy

g~

V

A

IV/AI

Axial ligand, L, in Mb(Fe 3 +)L

LS i

4-Melm 1-Melm Im 2-NH2Py 2-MePy 4-MePy Py 4-NH2Py 2-Melm none none

1.56 1.53 1.51 1.51 1.54 _ a 1.54 1.53

2.24 2.25 2.22 2.24 2.24 2.23 2.24 2.20 2.20 2.18 2.22

2.90 2.87 2.92 2.92 2.92 2.94 2.95 2.91 2.90 2.55 2.66

1.98 1.98 1.87 1.88 1.92

3.58 3.30 3.47 3.35 3.51

0.552 0.600 0.538 0.561 0.547

1.88 1.89

3.57 3.70

0.529 0.512

3.46 2.66

6.16 4.13

0.562 0.644

4-Melm l-Melm Im ° His(E7) His(E7) His(E7)? b His(E7) His(E7)[Im ] His(E7)[Im-]? b O H - and His(F8)[Im-] O H - and His(F8)[lm °]

LS b LS~

_

a

1.84 1.70

a Unobservable. b The axial ligand is not explicit.

values and line widths, their spectral features considerably differ from those of typical low-spin rhombic spectra. Therefore, we attempted spectral simulation of some Mb(Fe3+)-HALS spectra in order to estimate the line-width anisotropy and to obtain relevant g values. The method of simulation principally conformed to Isomoto et al.'s method [12] except that some modification was made for transition probabilities according to Aasa and V~innghrd [13] and the effect of line-width anisotropy was included in the calculation. The outline of the calculation is as follows. (i) The resonant field, H', is calculated with the following equations for all sets of (0, ~). H'( O, ok) = hu/flg

(7)

where o is the anisotropic line-width parameter expressed as o = ( 4 s i n Z 0 cos2~i, + Oyz sin20 sin2~ + o~cos20) '/2

(iii) The intensity, Ii, at the resonant field, Hi, is calculated by i, = Y' f , ( j -

,)p(e,,)

sin o,

O,q,

fori-m/2

< j
2

2

2

2

-2

2"~ I/2

g = (lxg x + lygy + lzg z j

(8)

where v is microwave frequency, fl is the Bohr magneton and l(lx, ly, lz) is a unit vector along the external field, H. (ii) Then, the following first derivative Gaussian shape function, f'(H), is introduced, f'(H)=

- [ ( H - H ' ) / o 3] exp[-(U-H')2/(2o2)]

(9)

(11)

where m is the number of the field division around H i, and P(8, if) is expressed as P ( O, ~ ) = ( g2 + g2y + g2 _ 1/g2( g~sin20 cos2~ + gy4sin2Osin2q~+ g~cos2O))/g

and

(10)

(12)

(iv) Finally,/i versus H i is plotted. Input parameters are as follows: (1) microwave frequency v, (2) principal g values (gx, gy, gz), (3) line width parameters (ox, Oy, oz), (4) division of (0, ~) plane, z~0 and A~ (0 ° ~< 0, ~ < 90°), and (5) division of the magnetic field, A H. Some of the resultant simulated spectra are shown in Fig. 4. The parameters obtained from the simulation calculation are listed in Table IV. Fig. 4

296

parameters, it is well understood that larger anisotropy in line width introduces larger deviation of spectra outline from the typical rhombic line space. Discussion

A

(3

B

b

I

I

I

0.1

0.2

0.3

I

I

0.4 0.5 BIT

I

I

I

0.6

0.7

0.8

Fig. 4. Observed (A, B) and simulated (a, b) spectra for Mb(Fe3+)(Py) (A and a) and Mb(Fe3+)(3-NH2Py) (B and b). Parameters used in the simulation calculation are listed in Table IV. indicates that the M b ( F e 3 +)-HALS spectra can be satisfactorily mimicked by the above calculation. C o m p a r i n g the simulated spectra with the best-fit

Table II shows that the small A values relate closely to give characteristic E P R features of the Mb(Fe3+)-HALS complexes. That is, a smaller ,~ introduces a larger spin-orbit interaction, leading to larger anisotropy of the g tensor. In addition, a weak axial field in these complexes induces the electronic states to approach the spin-crossover point, and the contribution of the excited states with different spin states to increase. Such an effect will result in the short spin-lattice relaxation time and cause the extreme line-width broadening. We consider, therefore, as pointed out in the previous paper [1], that stronger axial field perturbation, in general, leads to larger tetragonal splitting and smaller g anisotropy unless there is extensive geometric variation in the vicinity of haem iron. This can be well exemplified by the fact that the A values in Mb(Fe3+)-HALS are larger than those of H A L S in the ferric porphyrin system where the axial ligands are both the same as the exogenous ligands in the Mb(Fe3+)-HALS, where the haem iron is coordinated with the exogenous ligand and the histidine imidazole, which exerts a stronger ligand field than do the exogenous ligands. A m o n g the complexes of the Mb(Fe3+)-HALS series a good inverse correlation between A and the axial field strength, which is against the general expectation mentioned above, has been found, but such a correlation is not clear in the Mb(Fe3+)-HALS series. This m a y be attributed to the fact that the haem in ferrimyoglobin c o m -

TABLE IV BEST-FIT PARAMETERS USED IN THE EPR SPECTRAL SIMULATION OF FERRIMYOGLOBIN DERIVATIVES Definition of the parameters is given in the text. Complex

Mb(Fe 3+)(3-NH z PY) Mb(Fe3 +)(Py)

g values

line widths (10 4 T)

g~

gy

g~

~

°v

1.11 1.11

2.00 2.03

3.02 3.07

400 450

120 180

80 85

AO(°)

A,#(°)

A( 10-4 T)

1 1

3 3

10 10

297 plexes is mixed-liganded and the co-planarity of the ferric ion is not maintained among the series of the complexes. In addition, the globin part would have some influence on the axial ligation of the exogenous ligands, leading to breakdown of the systematic change of axial field strength or systematic displacement of the haem iron from the porphyrin plane by the exogenous ligands, as was discussed in the HALS series in the ferric porphyrin complexes. However, there seems to be an inverse correlation between z5 and I V/A I, as was found in the series of the ferric porphyrin-HALS complexes. For the pyrazole complexes, the principal axis system, 0ehich was assigned in the same manner as for the other ferric myoglobin or porphyrin systems, does not give the proper rhombicity, as shown in Table II. However, if the new axis system obtained by the rotation x-z, y - y and z - - x is used, the proper values are obtained. That is, 1.98, 3.36 and 0.588 for V, A and V/A, respectively, in Mb(Fe3+)Pz and 1.55, 3.68 and 0.423 for those in Fe3+(PP)(Pz)2, respectively. This means that the tetragonal axis in the pyrazole complex system does not coincide with the principal molecular axis and is in the porphyrin plane, contrasting with the common low-spin complexes of ferric porphyrins. Small but definite differences were observed among the A values of the L S i complexes for which histidine-E7 (His-E7) coordination has been assumed (see Table III). The LS i complex observed 4-NH2-pyridine system has larger A values than the others in the group. Though the A value for the 2-Me-imidazole system could not be determined directly from the EPR spectrum, its A value is estimated to be comparable to that of the 4-NH2-pyridine system from the similarity of the gy and g. values between the both systems. The L S i complexes with such larger A values will probably have histidine imidazoles whose N - H bond is strongly polarized, since those complexes are in a more basic condition than those of the rest of LS~, as is seen from the fact that the LS b complexes were concomitantly observed in the former system but were not observed in the latter. Walker et al. [14] and Satterlee et al. [15] have pointed out that Fe-Im interaction in haemoproteins may be significantly strengthened by weakening of the N - H

bond of the histidine. By the same consideration, we can trace the origin of the other two low-spin forms of ferrimyoglobin as follows. LS b may retain the hydroxide ion and the His-F8 with a strongly polarized N - H bond (Im-), while LS, may retain the His-F8 with a non-polarized N - H bond (Im°) and a water molecule with a partly polarized O-H bond. The smaller A in LS a than in LS b is well consistent with the above proposition that the weaker axial field will provide smaller tetragonal splitting. The H ÷ dissociation from the liganded imidazole of ferrimyoglobin complex above p H 9.0 has been observed by 1H-NMR by Iizuka et al. [16]. It seems quite interesting that M e O H and 4CN-pyridine enhance the denaturation to produce LS a. Probably, these compounds disturb the hydrogen bonding in the myoglobin molecule as the low-pH media do. This acidic form of ferrimyoglobin (LS a) in a low-spin form has not been identified hitherto, though a low-spin acidic form of horse heart ferricytochrome c has been reported and explained as a result of weakening of methionine-iron coordination through the 1HN M R experiment [17]. In conclusion, it turned out that the nitrogenous bases added to the ferrimyoglobin solutions as exogenous ligands brought about unique complexes with Mb(Fe 3÷) expressed as Mb(Fe 3+)(Lex ) and Mb(Fe3÷)-HALS, denatured ferrimyoglobin to produce Mb(Fe3÷)(Im[His-E7])expressed as LS i, Mb(Fe3÷)(OH-)(Im-[His-F8]) expressed as LS b, or Mb(Fe3÷)(OH-)(Im°[His-F8])expressed as LSa, or else isolated the haem from the globin part to produce characteristic low-spin HALS complexes.

References 1 Migita, C.T. and Iwaizumi, M. (1981) J. Am. Chem. Soc. 103, 4378-4381 2 Brautigan, D.L., Feinberg, B.A., Hoffman, B.M., Margoliash, E., Peisach, J. and Blumberg, W.E. (1977) J. Biol. Chem. 252, 574-582 30rme-Johnson, N.R., Hansen, R.E. and Beinert, H. (1971) Biochem. Biophys. Res. Commun. 45, 871-878 4 De Vries, S., Albracht. S.P.J. and Leeuwerik, F.J. (1979) Biochim. Biophys. Acta 546, 316-333 5 Carter, K.R., Tsai, A. and Palmer, G. (1981) FEBS Lett. 132, 243-246

298 6 Dickinson, L.C. and Chien, J.C.W. (1977) J. Biol. Chem. 252, 1327-1330 7 Caughey, W.S. (1966) in Hemes and Hemoproteins (Chance, B., Estabrook, R.W. and Yonetani, T., eds.), pp. 276-281, Academic Press, New York 8 Blumberg, W.E. and Peisach, J. (1971) in Probes of Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T. and Mildvan, A.S., eds.), Vol. II, pp. 215-229, Academic Press, New York 9 Hori, H. (1971) Biochim. Biophys. Acta 251,227-235 10 Mailer, C. and Taylor, C.P.S. (1972) Can. J. Biochem. 50, 1048-1055 11 Taylor, C.P.S. (1977) Biochim. Biophys. Acta 491, 137-149

12 Isomoto, A., Watari, H. and Kotani, M. (1970) J. Phys. Soc. Japan 29, 1571-1577 13 Aasa, R. and V~inngMd, T. (1975) J. Magn. Resonance 19, 308-315 14 Walker, F.A., Lo, M.-W. and Ree, M.T. (1976) J. Am. Chem. Soc. 98, 5552-5560 15 Satterlee, J.D., La Mar, G.N. and Frye, J.S. (1976) J. Am. Chem. Soc. 98, 7275-7282 16 lizuka, T. and Morishima, I. (1975) Biochim. Biophys. Acta 400, 143-153 17 Morishima, I., Ogawa, S., Yonezawa, T. and Iizuka, T. (1977) Biochim. Biophys. Acta 495, 287-298