Structural Models for Iron and Copper Proteins Based on

17 Structural Models for Iron and Copper Proteins Based on Spectroscopic and

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Magnetic Properties HARRY B. GRAY California Institute of Technology, Pasadena, Calif. 91109

The spectroscopic and magnetic properties of several iron(III) model systems have been investigated and compared with those of certain iron-containing proteins. Spectral data show that [Fe(III)O6] octahedral coordination is present in the Saltman-Spiro iron(III) polymer and probably also in the ferritin core. A tetrahedral polymeric [Fe(III)O ] coordination is compatible with the spectral and magnetic data found for iron(III) phosvitin. Extensive data have been collected on oxobridged Fe(III) dimers. An electronic structural model involving a moderate antiferromagnetic coupling of the two high-spin Fe(III) centers satisfactorily explains the ligand field spectra. Evidence for simultaneous pair electronic excitations is also presented. The spectral and magnetic data for hemerythrins are interpreted in terms of a dimeric Fe(III) model, and a general two-electron oxidative addition binding model is proposed for oxyhemerythrin, oxyhemoglobin, and oxyhemocyanin. 4

" C o r several years my coworkers and I have been studying the electronic absorption spectra of models for metal proteins and, i n certain cases, of the proteins themselves. In favorable cases, additional information has been gathered from infrared spectral and magnetic susceptibility measurements. Our objective i n this work is the development of a line of attack which w i l l allow the construction of a reasonable model for the structure in the immediate vicinity of the metal, or the coordination structure, of the metal protein. 365 In Bioinorganic Chemistry; Dessy, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1971.


366

BIOINORGANIC CHEMISTRY

Figure 1. Refotionship of the lowest LF energy levels for d Fe(III) complexes in octahedral and tetrahedral coordination 5

Figure 2. Electronic absorption spectra of Fe(H 0) in ferric ammonium sulfate and [Fe(/I/X> ] in orthoclase feldspar 2

4

3+

6

iei

In Bioinorganic Chemistry; Dessy, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1971.

17.

Iron and Copper Proteins

G R A Y

High-Spin

Pe (III)

367

Complexes

A large part of our effort has been directed toward an understanding of the electronic spectra of d high-spin complexes of the structural types [ F e ( I I I ) 0 ] o c t and [ F e ( I I I ) 0 ] t e t . The interesting situation with re spect to the ligand field ( L F ) transitions for these two types of com plexes is shown i n Figure 1. There are no spin-allowed L F transitions from a A i ground state, and the two lowest spin-forbidden transitions decrease i n energy as the cubic L F splitting increases. Therefore, the first two spin-forbidden L F bands for [ F e ( I I I ) O ] o c t should occur at lower energies than the analogous bands for [ F e ( H I ) 0 ] t e t . 5

6

4

6


e

4

Experimental spectra of two structurally well-defined model systems illustrate this difference ( I ) . The upper spectrum of Figure 2 is that of a single crystal of ferric ammonium sulfate which contains F e ( H 0 ) ; the lower spectrum is that of an Fe -doped sample of orthoclase feldspar. In this sample, F e ions replace A l i n a certain percentage of tetra hedral oxide donor sites. The two spectra show very clearly that the two lowest bands, Α χ ^ ^ G ) and Α χ - » T ( G ) , are substantially lower in energy in the [ F e ( I I I ) 0 ] o c t case. These spectra were selected because four L F bands are resolved in each case. The third band is fairly sharp in each spectrum and is assigned to the transition which does not require orbital promotion, Α χ - » A i , E ( G ) . The fourth band i n each case is assigned A i T ( D). 2

6

3 +

3+

3 +

3 +

6

4

6

4

2

6

6

4

6

4

4

4

4

2

Summaries of the spectral data for F e ( H 0 ) and [ F e ( I H ) 0 ] e t are given in Tables I and II, respectively. The molar extinction coeffi cients of the spin-forbidden L F bands are relatively small, as expected. The bands for [ F e ( I I I ) 0 ] t are approximately a factor of ten more intense than those for F e ( H 0 ) because the L F transitions in the latter complex are both spin- and orbitally-forbidden. The computed energy 2

4

4

6

3 +

Electronic Spectral D a t a for F e ( H 0 ) e Fe (S0 ) · (NH ) S0 · 24H O 2

2

4

3

4

v,

4

T

2

(A 4

4

T

2

1;

«E)

t

t e

2

Table I.

3 +

6

2

4

2

3 +

in

a

ε

cmr

16

12,600

0.05

18,200

0.01

24,200 24,600 25,400

1.3

27,700

1

From Réf. 1. The spectrum may be fit satisfactorily using the following parameter values: lODq(oct) = 13,700 cm" ; C/B = 3.13; Β = 945 cm- . a

b

1

1


368


Table II.

Electronic Spectral Data for [Fe(III)0 ] in Orthoclase Feldspar 4

0

v, c m

Ti T ( A T 2

4

4

α

4

E)

2

Calcd

b

22,150 24,550 26,550 28,940

From Réf. 1. For A(tet) = 7350, Β = 540, C = 4230 cm" . 1


b

lf

0.73 0.76 4.1 0.1

22,500 23,900 26,500 29,200

4

4

ε

- 1

Dq/B

Figure

3.

Calculated LF energy levels for [Fe(III)O -] 6

0Ct

level diagrams shown i n Figures 3 and 4 are based on the experimental data for the two model complexes. T h e value of is considerably larger than A t , i n agreement with expectation. O f interest is the large difference i n the Racah parameter ratio C / B — f o r the octahedral case, this ratio is approximately 3, whereas for the tetrahedral coordination it is about 8. te



17.

G R A Y

369


20 h

1

υ

Figure

4.

Dq/B

2

Calculated LF energy levels for [Fe(III)0,] tet

The Ferritin

Core

A n interesting biological system which contains F e in an [ F e ( I I I ) O ] coordination structural environment is the iron-storage protein, fer ritin. The core of ferritin consists of a phosphate-containing, iron ( H I ) hydrated oxide or oxide-hydroxide (2). Models have been suggested for the ferritin core which feature F e ( I I I ) in [ F e ( I I I ) 0 ] t e t (3), [ F e ( I I I ) 0 ]oct (4), and mixed [ F e ( I I I ) 0 ] t e t - [ F e ( I I I ) O e ] o c t (5) coordination environments. 3+

w

4

6

4

A particularly interesting synthetic model for the ferritin core is the iron (III) polymer obtained by Spiro, Saltman, and coworkers from b i carbonate-hydrolyzed ferric nitrate solutions (6). The polymer has an approximate molecular weight of 150,000 and is about 70 A i n diameter. Analytical studies indicate a composition F e 0 3 ( O H ) 4 ( N 0 ) 2 · 1 . 5 H 0 for the polymer. In physical size and shape, the Saltman-Spiro ball is remarkably similar to the ferritin core. The structure that has been sug4

3


2

370


gested (3) for the Saltman-Spiro ball and implied for ferritin is shown i n Figure 5. T h e F e ( I I I ) ions are bound together i n a network of bent oxo and hydroxo bridges with a tetrahedral coordination structure, [ F e ( I I I ) 0 ] t e t . I n this model, water molecules and N 0 " ions coat the ball, leading to Saltman's graphic analogy with M and M's candy ( 7 ) . The principal basis for the [ F e ( I H ) 0 4 ] t t coordination structural model is a low-angle x-ray scattering experiment and subsequent analysis of the radial distribution function ( 3 ) . This is a difficult experiment to analyze, and i n our opinion electronic spectroscopy offers a more foolproof route to the coordination structure. 3

4


e

Biochemistry

Figure 5. Structural model proposed for the Saltman-Spiro ball, adapted from Ref. 3 W e have examined ( 8 ) , as d i d Brady and coworkers ( 3 ) , the elec tronic absorption spectrum of the Saltman-Spiro ball. The spectrum ( F i g ure 6) (S) shows a pattern of four weak bands with the lowest band at about 900 nm, i n very good agreement with [ F e ( I I I ) 0 ] o c t coordina tion. The derived L F parameters are = 11,260 cm" , C / B = 3, and Β = 815 cm" . W e can say with considerable confidence that most of the F e ( I I I ) ions occupy octahedral coordination sites. There is no hint of bands attributable to [ F e ( I I I ) 0 4 ] t , and as these bands are intrin sically more intense than are those assigned to [Fe(III)Oe]oct, we can effectively rule out tetrahedral coordination i n this synthetic model compound. 6

1

1

te



17.

371


G R A Y

o.o L i 200

ι

ι

ι

ι

400

600

800

1000

i_ 1200

X(nm)

Figure 6.

Electronic absorption spectrum of the SaltmanSpiro ball

Our electronic absorption spectral studies on the ferritin core itself are less definitive (8, 9 ) . Because of strong tail absorption through the visible, we have been able to resolve only a 918-nm L F band. This band is diagnostic of [ F e ( I I I ) 0 ] o c t coordination, but without better spectra in the 400-700-nm region we cannot distinguish between exclusively [Fe(III)O ]oct and mixed [ F e ( I I I ) 0 ] o c t - [ F e ( I H ) 0 4 ] t e t coordination structures. There is, however, no positive evidence for the presence of [ F e ( I I I ) 0 ] t e t units i n the core and the analogy with the Saltman-Spiro ball leads us to prefer a model with [ F e ( I I I ) 0 ] o c t coordination. Another important structural feature in both the Saltman-Spiro ball and the ferritin core that can be examined by spectroscopic means is the question of whether the polymeric F e ( I I I ) network is held together through both oxo and hydroxo bridges, as suggested i n Figure 5, or whether oxo (or hydroxo) bridges alone suffice. Infrared spectral stud ies of various hydrated iron oxides of known structure have shown that bridging hydroxide invariably absorbs i n the 900-1200-cm" region (9). Neither the Saltman-Spiro ball nor the ferritin core shows an infrared band attributable to bridging hydroxide (9), although absorption owing respectively to N 0 " and P 0 ' in the region of interest casts some doubt on the conclusion. It seems highly probable, however, that both the Saltman-Spiro ball and the ferritin core contain disordered polymeric networks of [ F e ( I H ) 0 ] o c t units primarily finked by bent oxo bridges. 6

e

6

4

6

1

3

4

3

6


372


Presumably, throughout these polymeric networks water molecules and N 0 " (or PO4 ') are bound to some of the F e ( I I I ) centers. 3

3

Iron (III)

Phosvitin

Another iron (III) protein where [ F e ( I I I ) O ] coordination is likely is iron (III) phosvitin. The fact that 46 F e ( I I I ) ions are bound b y one molecule of phosvitin has recently been established b y Saltman and Multani (10). Electronic absorption spectra of the iron (III) protein are shown i n Figure 7 ( I I ) . Three weak L F bands are resolvable i n the 150°K spectrum, at 447, 426, and 400 nm. The relatively high energies of the first two L F bands suggest [ F e ( I I I ) 0 ] t e t coordination, and i n fact a very satisfactory fit of the spectrum is obtained with the L F pa rameters A — 5270 cm" , Β = 495 c m , and C / B = 8. Notably, the spectrum is apparently devoid of peaks in the 800-1000-nm region where [Fe(III)O ]oct invariably appears. Thus, we suggest that iron (III) phos vitin provides a biological example of the fairly rare [ F e ( I I I ) 0 ] t e t coordination. T w o more bits of information tempt us to build up the detail of the model. W e have measured the magnetic susceptibility of iron (III) phos vitin over the temperature range 8 5 ° - 3 0 0 ° K and find evidence for antiferromagnetic behavior (Figure 8) ( I I ) . The magnetic moment per iron drops from 4.45 to 3.39 B . M . over the temperature range investigated. Thus, it is highly probable that at least some of the [ F e ( I I I ) 0 ] t units are interacting through ligand bridges. As there are 100 serine phos-


n

4

1

t e t

-1

e

4

4

0.8

t e

Visible and Near I.R. spectrum: Iron-Phosvitin (8.675 mg/ml, pH 7.5, 0.005 M Tris) III

380

420 460 Wavelength (hm)

500

Analysis: bands at 447, 426, 400 nm with C/B = 8.0 (T value) rf

Β = 495 cm- Dq = 527 cm>—ι 1

400

600

800

1000

Wavelength (nm)

Figure 7.

Electronic absorption spectra of iron(III) phosvitin


17.

373


G R A Y

Temperature-Dependence of magnetic moment of Iron-Phosvitin 5.0

î

3.39 B . M 4.0 • (85°K)

«

4.45 B . M . (300°K)

S φ

I: Kambe theory, Si = 5/2, S = 5/2. J = -12 cm-

s

II: Iron-Phosvitin

2

Ι 3.0

1


"-5

i

2

0

1.0

_1_

200

100

300

Temperature, °K

Figure 8. Magnetic moment data for iron(III) phosvitin

- ρ

I

ι Fe

Fe

.Fe -P

%

Ρ

Α

ι Fe

Oi

/Fe P-

Ρ

Figure 9. Model for iron(III) binding by phosvitin phate residues i n phosvitin, or roughly two for each F e ( I I I ) , an inter esting possibility is a structure of the type shown i n Figure 9. A number of other models are possible, of course. F o r example, a small percentage of octahedrally coordinated F e ( I I I ) ions could escape detection i n our spectroscopic experiment. W e should emphasize the major conclusions: Most of the F e ( I I I ) ions are tetrahedrally coordinated and form some type of polynuclear structure. Oxobridged Iron (III) Dimer s Our structural investigations of hydrolytically polymerized F e ( I I I ) have not been limited to the high molecular weight systems such as the Saltman-Spiro ball. M u c h more of our research, i n fact, has been con centrated i n the area of relatively simple binuclear F e ( I I I ) complexes. The polyfunctional ligands E D T A (ethylenediaminetetracetate) and


374


H E D T A ( hydroxyethylethylenediaminetriacetate ) chelate F e ( I I I ) so effectively that upon base hydrolysis only one coordination position is available for bridging, and dimers of the type ( E D T A F e ) 0 " and ( H E D T A F e ) 0 " , but no higher polymers, form (12, 13, 14). As a result, E D T A and H E D T A are excellent solubilizing agents for F e ( I I I ) over a wide p H range extending to very basic solutions. 2

2

Red crystalline salts can be obtained from the red solutions containing the dimeric complexes (13). In particular, we have investigated the enH (ethylenediammonium) salt of ( H E D T A F e ) 0 " . The structure of this complex is known from x-ray crystallographic analysis (15). The most important features of the coordination structure are shown i n F i g ure 10. The monomelic units [ F e ( I I I ) 0 N ] o c t share a nearly linear oxo bridge, the F e ( I I I ) - 0 - F e ( I I I ) angle being 165°. The Fe(III)-to-oxo distance is 1.8 Â, a number which might be considered an indication of ρπ —» dm bonding were it not for the fact that a similarly short distance (1.68 A ) has been observed i n an A l ( I I I ) - 0 - A l ( I I I ) structure (16). 2


4

2

2 +

2

4

2

2

The ( H E D T A F e ) 0 " complex possesses an infrared absorption band at about 840 cm" which is characteristic of an almost linear Fe( III ) O - F e ( I I I ) unit (13,14, 17, 18). This spectral feature of the complex is shown in Figure 11 (18). The band is assigned to the antisymmetric stretching motion i n the three-atom system. The fact that the 840-cm' band is present both i n a crystal and i n a D 0 solution of ( e n H ) [( H E D T A F e ) 0 ] · 6 H 0 establishes that the oxobridged structure per sists in aqueous media. Magnetic measurements and electronic spectral 2

2

1

1

2

2

2

Ν

Figure 10. Coordination structure of (HEDTA Fe) 0 ~ 2

2


2

17.

G R A Y

375

Iron and Copper Proteins enH [(FeHEDTA) 0] · 6H 0 2

2

solution in D 0 solid ~ 300°K

solid ~ 100°K


2

I I 900 800 ν (cm ) -1

Figure 11.

I I 900 800 ν (cm )

2

I 1/ I 900 800 ν (cm )

-1

-1

Infrared spectra of (HEDTA in the 840-cm' region

Fe) ÇP~ 2

1

6.0 [Fe(pic) (OH )Cl] 2

2

5.0

4.0 H 3.0

H 2.0 H

[enH,][(FeHEDTA) 0] · 6H,() 2

1.0

0 20 40 60 80 100 120 140 160180200220 240 260 280 300 Temperature, °K

Figure 12. Magnetic moment data for (enH )[( HEDTA FefG\ - 6H 0 and [Fe(pic XH 0)Cl'] (pic = picoUnate) 2

2

2

2

data also confirm the essential identity of the structure i n solids and aqueous solutions (18). Turning to the magnetic data, Figure 12 shows /x.ff vs. Τ plots for a solid sample of ( e n H ) [ ( H E D T A F e ) 0 ] · 6 H 0 , and a reference highspin (S — 5/2), monomeric F e ( I I I ) complex. The binuclear complex exhibits antiferromagnetic behavior, the fi t per F e ( I I I ) dropping from 1.9 B . M . at 300°K to effectively zero at about 30°K. A n excellent fit of these data is produced by assuming that two S = 5/2 ions are spin coue

2

2

2

ef


376


pled, with J = —95 cm" . Very strong supporting evidence for this model is provided by the electronic spectral data to be discussed later. 1

Examination of the literature i n this field reveals (18) that oxobridging is well established i n several binuclear F e ( I I I ) complexes. The oxobridged F e ( I I I ) complexes which have been carefully studied all exhibit / values i n the range —85 to —105 cm" . In sharp contrast, the one dihydroxobridged F e ( I I I ) dimer that has been isolated and fully characterized, [ F e ( p i c ) O H ] , displays a much weaker spin-spin cou1

2


pling [/ =

-8

2

cm" , /xeft ( 3 0 0 ° K ) — 5.2 B . M . ] 1

10 v, cm -3

Figure 13.

(17).

-1

Electronic absorption spectrum of (HEDTA Fe) 0 ~ 2

2

Virtual proof that ( H E D T A F e ) 0 " contains antiferromagnetically coupled octahedral S = 5 / 2 units may be extracted from its electronic absorption spectrum, which is shown i n Figure 13 (18, 19). H E D T A is a good ligand for this study because it does not absorb appreciably i n the near IR, visible, and near U V . N o fewer than eight absorption peaks appear before intraligand and charge transfer absorption take over. The spectrum divides into two parts, a set of four low-energy peaks a-d with low to moderate extinction coefficients, and a group of more intense, higher-energy bands (e-h). 2

2

Bands a-d are positioned properly to be the first four L F transitions in each S = 5/2 [ F e ( I I I ) 0 4 N ] o c t unit (see Figure 3 ) . Reasonable 2


17.

G R A Y

377


agreement between theory and experiment is obtained for the L F pa rameter values Δ** = 10,900 cm" , C / B == 2.4, and Β — 950 cm" . The main difference between the L F spectrum in the oxobridged dimer and an octahedral monomeric F e ( I I I ) complex is in the intensities of the bands. The molar extinction coefficients of the four L F bands in ( H E D T A F e ) 0 " , for example, are two orders of magnitude larger than those for a monomeric complex such as F e ( H 0 ) . This intensity en hancement of the spin-forbidden L F bands is an expected consequence of the antiferromagnetic coupling in the oxobridged system; the coupling partially relaxes the spin selection rule and the transitions acquire some allowed character. The relationship of the spin-spin coupling to the intensity enhancement of spin-forbidden L F bands has been worked out in some depth in the case of M n F and other M n ( II)-containing antiferromagnetic materials (20, 21). Our observation in the F e ( I I I ) - 0 F e ( I I I ) system logically has the same explanation. 1

1

2

2


2

6

3 +

2

The four relatively intense U V bands (e-h) puzzled us for a long time. Based on our studies of related monomeric complexes, energetically they are too low to be attributable to ligand - » F e ( I I I ) charge transfer transi tions; what is more, there are too many bands for a charge transfer expla nation. The bands are much too intense to be the higher-energy L F bands, even after allowing for the intensity enhancement in an antiferromagnetically coupled dimer. Elimination of the usual types of transitions has led us to suggest that the U V spectrum consists of simultaneous pair electronic ( S P E ) excitations (19). That is, simultaneous L F transitions on the two F e ( I I I ) centers can be coupled so that the pair excitation is spin-allowed. This explanation is not without precedent—Varsanyi and Dieke first pro posed (22) a similar explanation for a portion of the fluorescence excita tion spectrum of P r C l , and for this system Dexter has discussed (23) S P E excitations from a theoretical point of view. Such transitions in mixed M n ( I I ) - N i ( I I ) fluorides (24) and dimeric C u ( I I ) acetate (25) have also been suggested in interpretations of absorption spectra. 3

Assuming simple additivity, the positions of four S P E excitations in ( H E D T A F e ) 0 " agree extremely well with the positions of bands e-h and provide considerable support for the interpretation. This agreement is shown in Figure 13 and is included in a summary of the interpretation of the electronic absorption spectrum of ( H E D T A F e ) 0 " set out in Table III. 2

2

2

2

In summary, we are well on our way to an understanding of the spectral and magnetic properties of certain types of binuclear F e ( I I I ) complexes. Oxobridged dimers of high-spin F e ( I I I ) monomers show at least four distinctive characteristics: A n infrared band around 840 cm" ; antiferromagnetic behavior with / in the range —85 to —105 cm" ; L F 1

1


378


Table III. Electronic Spectral Data for enH [(HEDTA Fe) 0] · 6 H O 2

2

Band


a b c d e f g h

2

Assignment

(10- ) v x , em-Ke) 3

m a

11.2 18.2 21.0 24.4 29.2 32.5 36.8 42.6

(2.6) (40) (25) (120)

a

•A, - » •Ai A -» a + b α + c 6 + 6 6 + d 6

t

"T ( A„ «E) "T = 29.4 = 32.2 = 36.4 = 42.6 2

4

2

° From Ref. 19. bands with enhanced intensity; and a rich series of U V bands, which we have interpreted as S P E excitations. Hemerythrin The spectroscopic and magnetic properties observed for the model oxobridged dimer, ( H E D T A F e ) 0 " , have proved of value in attempts to understand the coordination structure of the protein hemerythrin, which binds oxygen reversibly. The protein from the sipunculid Golfingia gouldii has been studied extensively by Klotz and coworkers (26). Eight subunits, each containing two iron atoms capable of binding one oxygen molecule, associate in the full protein to give a molecular weight of about 108,000. Magnetic measurements show that the deoxy form of the pro tein contains high-spin F e ( I I ) (26). The electronic absorption spectrum of this form is relatively clear down to 300 nm; in the U V , the peak at about 280 nm characteristic of tyrosine appears. 2

2

Upon oxidation to the met [ F e ( I I I ) ] protein, absorption peaks ap pear in the visible region. Each subunit of the met protein binds ligands such as CI", Br", and Ν»" in a 1:2 F e ( I I I ) complex, and the visible peak positions of these met derivatives vary slightly (27). The electronic ab sorption spectrum of metchlorohemerythrin is shown in Figure 14 (28). In addition to the bands shown, there is some evidence for a peak owing to an electronic transition in the 900-nm region (28). This peak is heavily overlapped by bands attributable to vibrational overtones and is thus not well characterized. The bands at 668 and 500 nm, however, may be assigned as L F bands in an F e ( I I I ) complex. Their moderate intensities (27, 28) are consistent with a model in which two F e ( I I I ) ions are spincoupled in an oxobridged dimer of the type [ C l - F e ( I I I ) - 0 - F e ( I I I ) ] . Magnetic susceptibility data to be discussed momentarily support this proposal.



17.

G R A Y

379


Figure 14.

Electronic absorption spectrum of metchlorohemerythrin

The bands at 384 and 331 nm i n the metchloro derivative must also be attributable to some kind of electronic transition involving one or both of the F e ( I I I ) ions. Their positions and intensities can be rationalized if they are assigned to S P E excitations i n an oxobridged dimer. A l ternatively, ligand F e ( I I I ) charge transfer transitions are also possible explanations. A t wavelengths lower than 331 nm, the tyrosine absorption takes over and additional peaks owing to F e ( I I I ) are presumably buried. The electronic absorption spectrum of oxyhemerythrin is shown i n Figure 15 (28). This spectrum closely resembles that of metchlorohemerythrin, except for the much greater intensity i n the band at about 500 nm. The spectrum can be considered as reasonable proof of a suggestion first made by Klotz (29) that i n the oxy form both irons have been oxidized to F e ( I I I ) and the 0 reduced to 0 " . Large intensity at 501 nm can be easily understood i n this model because a similar absorption band has been observed in the complex formed between [ F e ( I I I ) E D T A ] " and H 0 i n basic solution (30). Presumably, this band is attributable to an 0 " - » F e ( I I I ) or H O O " -> F e ( I I I ) charge transfer transition. 2

2

2

2

2

2

2


380



20

200

300

400

500

600

700

800

900

X(iim)

Figure 15.

Electronic absorption spectrum of oxyhemerythrin

The bands at 370 and 317 nm i n oxyhemerythrin are similar to the 384 and 331 nm bands i n the metchloro derivative and i n all probability analogous assignments are appropriate. The spectral data for the metchloro and oxy proteins suggest coordination structures involving dimeric F e ( I I I ) units. Magnetic susceptibility experiments over a limited temperature range for both proteins add considerable weight to this suggestion. These data are shown i n Figure 16 (28). Although the error limits are large, it is apparent that i n each case the ^ ff per F e ( I I I ) is greatly reduced from the high-spin value of ~ 6 B . M . Furthermore, i n the range 300° to 100°K there is some evidence that both proteins exhibit antiferromagnetic behavior. e

The spectral and magnetic data point to an oxobridged F e ( I I I ) dimer structure for the metchloro protein. A n oxobridged model is also attractive for the oxyhemerythrin, but the alternative possibility of a


17.

381


G R A Y

h6BM


Oxyhemerythrin (—)

Met (—-)

r-2BM

3Ô0K

IJK>K

Figure 16.

Magnetic moment data for metchlorohemerythrin hemerythrin

and oxy-

Fe(n0-O-FednKX)H He

Fe binding by hemoglobin are shown in Figure 18. The classic Pauling model ( 1936 ) involves delocalized bonding of 0 to low-spin F e ( I I ) , with a bent F e - O - O unit (32). In 1956, Griffith pointed out that the bonding i n oxyhemoglobin could be formulated along the same lines as i n metal-ethylene π-complexes and suggested a model in which 0 bonds symmetrically to a low-spin F e ( I I ) (33). 2

2

A third model, involving F e ( I I I ) and 0 " , aroused some controversy after it was proposed by Weiss in 1964 (34, 35, 36, 37, 38), but it has recently been supported by Peisach and coworkers (38). This binding model for oxyhemoglobin has become particularly attractive i n view of the structural data now available on 0 binding to C o (II) complexes. In a number of well established cases (39, 40, 41, 42), including the very interesting cobalt hemoglobin (or coboglobin) (43), 0 is reduced to 0 " and cobalt is oxidized to C o ( I I I ) , so the mode of 0 binding is 2

2

2

2

2



17.

383


GRAY

Low-spin d*- 0

Figure 18.

Spin-coupled d - O, 5

2

Four models for 0 -binding in oxyhemoglobin 2

C o ( I I ) + 0 — [ C o ( I I I ) ( 0 - ) ] . Furthermore, the C o ( I I I ) - 0 - 0 unit is bent. In the [ F e ( I I I ) - ( 0 " ) ] model, the diamagnetism of oxyhemoglobin must be explained by postulating (38) an appropriately large antiferro magnetic coupling between low-spin F e ( I I I ) and 0 ~. Although Peisach and coworkers (38) cite some spectral evidence in support of the pres ence of F e ( I I I ) , the strong analogy between met and oxy derivatives established from magnetic and spectral data for hemerythrin is lacking. Furthermore, there is one serious objection to the ' metsuperoxide" model. Our spectral studies of several model C o (III) complexes containing 0 ~ show that this ligand falls very close in the spectrochemical series to — N C S " (44). Met thiocyanate hemoglobin has considerable high-spin (S = 5/2) character at room temperature (45), whereas at this tem perature oxyhemoglobin has no unpaired electrons. O n the basis of this correlation, it is difficult to understand why only low-spin F e ( I I I ) should be present in an 0 "-bound product. W e have tried without success to locate the Ο—Ο stretching fre quency which would be expected in the infrared spectrum for an unsymmetrically bound 0 or 0 ~ in oxyhemoglobin. W e have measured (31) infrared spectra for oxyhemoglobin and carbonylhemoglobin in the region 2

2

2

2

2

2

2

2


384


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Τ" ι

-1

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D 0^

H0

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\

D0 2

\ ι 0

Figure 19.

CO 20

o2

2

40

— H b -o 2 — Hb-

1 11 \J /

60

t

ΛΛ


200

Structural Models for Iron and Copper Proteins Based on

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