A spectral study of human ceruloplasmin

132

Biochimica et Biophysica Acta, 534 (1978) 132--140

© Elsevier/North-Holland Biomedical Press

BBA 37897 A SPECTRAL STUDY OF HUMAN CERULOPLASMIN

SHMUEL FREEMAN and EZRA DANIEL Department o f Biochemistry, The George S. Wise Center for Life Sciences, Tel-Aviv University, Te l-A viv (Israel)

(Received September 13th, 1977)

Summary The absorption, luminescence and CD spectra of human ceruloplasmin were studied. The absorption spectrum in the infrared, and the CD spectrum in the ultraviolet, both indicate the presence of fl conformation in the native structure of the protein. From the magnitude of the measured ellipticity, it is estimated that 0.46 of the amino acid residues are in the fl conformation, the remaining 0.54 being in an unordered form. A comparison of the fluorescence and phosphorescence properties of native and apoceruloplasmin shows that the presence of copper causes the quenching of t r y p t o p h a n y l luminescence, probably through energy transfer to the copper chromophores. By the combined resolution of the absorption and CD spectra, it was concluded that the copper chromophores are involved in six electronic transitions in the region 300-900 nm. Our results provide evidence for an interaction between the copper chromophores responsible for the 330 nm absorption in ceruloplasmin.

Introduction

Ceruloplasmin is a copper-containing protein present in the plasma of vertebrates. The copper is believed to be involved in the oxidase activity of the molecule. As a consequence, effort has been deployed to determine the number of copper atoms per molecule, their oxidation state, and their contribution to the magnetic and optical properties of the native protein. For some time, it was accepted that ceruloplasmin contains eight copper atoms per molecule [1]. Recent molecular weight determinations [ 2--4], however, imply that the number of copper atoms has to be less than eight. An EPR study of nitric oxide-treated reduced ceruloplasmin [5] indicated the presence of dipole-dipole coupled copper ion pairs. Indirect evidence for pairing comes from the finding that part of the copper in ceruloplasmin is EPR non
133 can accept equals the number of copper atoms present [6,7]. In this communication, we report on a study in the course of which the absorption, luminescence and circular dichroic properties of human ceruloplasmin were investigated. Our results provide corroborative evidence for the pairing of copper in ceruloplasmin. Experimental Human ceruloplasmin, prepared by the method of affinity chromatography, was obtained from Miles Yeda, Rehovoth. Its properties were described in a previous report [4]. Protein concentrations were determined spectrophotometrically, using ~1~ = 14.9 at 280 nm [8] Apoceruloplasmin was pre~1 am pared by exhaustively dialyzing a 10 mg/ml solution of ceruloplasmin against 0.05 M KCN in 0.1 M sodium phosphate buffer, pH 7.2. The cyanide was subsequently removed by dialysis against buffer. Complete removal of copper was ascertained by atomic absorption measurement. Absorption measurements were carried out with a Cary 14 or Zeiss PMQ II spectrophotometer. Infrared spectra were measured with a Perkin-Elmer 457 A grating infrared spectrophotometer. To prepare the solid film, a solution of the protein (10 mg/ml in 0.1 M phosphate buffer, pH 7.2) was deposited on a crystal of thalium iodide-bromide and evaporated to dryness at room temperature. CD spectra were recorded with a Durrum-Jasco spectropolarimeter, Model J-10, as described elsewhere [9]. In the calculation of the mean residue ellipticity a mean residue weight of 115, calculated on the basis of the amino acid composition of ceruloplasmin [8], was used. Except for the wavelength span extending from 300 to 400 nm, the absorption and CD spectra in the visible light were resolved by simultaneous fitting with a minimal number of Gaussian bands, such that each component band had the same location and bandwidth in both CD and absorption [10,11]. Fluorescence and phosphorescence spectra (uncorrected) were measured with a Hitachi-Perkin-Elmer spectrofluorophotometer, Model MPF-2A. The experimental details used in the determination of phosphorescence spectra and phosphorescence lifetimes were as described elsewhere [12]. Determination of copper content was made with a Varian Techtron model AA-5 atomic absorption spectrometer, using an airacetylene flame. A control experiment with purified bovine serum albumin showed that background absorption by protein was negligible. Results

Copper content Native ceruloplasmin was found by us to contain 0.257% copper. This determination of the copper content, taken in conjunction with a molecular weight of 124 000 [4], gives the number of copper atoms in the molecule as 5.1. These values are somewhat lower than the corresponding ones of 0.275% and 5.7 reported by Magdoff-Fairchild et al. [2]. Absorption The difference absorption spectrum of native and apoceruloplasmin, showing

134

~k, YIFfl 300

400

500

600

700

800

1.2

E

0.8

o

L.

o

U ~" 0.6 v.X ~ 0.4

0"2 I 0

I 3.0

2.5

2.0 ~'X10-4

1.5

cm-1

Fig. 1. D i f f e r e n c e a b s o r p t i o n s p e c t r u m o f h u m a n n a t i v e a n d a p o c e r u l o p l a s m i n . T h e o r d i n a t e r e p r e s e n t s the molar extinction coefficient, calculated on the basis of the total copper content of ceruloplasmin. O b s e r v e d v a l u e s ( o ) a r e r e a d i n g s a t i n t e r v a l s o f 1 0 n m o f t h e r e c o r d e d s p e c t r u m . T h e solid line ( ) is the calculated sum of constituent bands of Gaussian shape (...... ) obtained by combined resolution of both absorption and CD spectra. Conditions: phosphate buffer, ionic strength 0.1, pH 7.2, 25°C.

I

I

---I . . . .

80

Z

60

1

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E

4o

c

20

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0 2000

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1400

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1200

135 TABLE

I

PARAMETERS OF COMPONENT BANDS OBTAINED AND CD SPECTRA OF HUMAN CERULOPLASMIN Results are measured

in phosphate

Wavenumber of extxemum ( v , c m -1)

Half bandwith ( c m -1)

31 22 18 16 14 12

5345 1653 1828 1318 1405 1083

000 000 350 260 000 250 * ** *** t tt

IN THE RESOLUTION

OF THE ABSORPTION

buffer, I = 0.1, pH 7.2, and 25°C. Absorption

bands *

Rotatory

bands *

** e (cm 2/mmol)

Oscillator strength * * *

482 183 471 1186 518 277

0.020 0.002 0.007 0.012 0.006 0.002

(eL~R) (cm 2/mmol)

0 ---0.606 0.595 0.333 --0.537 --tt

Rotational strength t X 1040 ergs • cm 3 • tad 0 --1.86 2.42 1.10 --2.20 --it

Calculated on the basis of the total copper content of the protein. Half-width at e/e or (eL--eR)/e. Calculated from the equation [39]: f = 4.32 • 10-9fe(P)dp. Calculated from the equation [19]; R = 22.9 Due to limitations of the instrument the CD measurements could not be extended determination of the rotatory parameters of this band.

lO"40f[(eL--eR)/V]dv.

to permit

the

the c o n t r i b u t i o n of the copper c h r o m o p h o r e s is presented in Fig. 1. By comparison with the corresponding CD spectrum, the absorption spectrum was resolved into six Gaussian bands with the parameters shown in Table I. T he infrared absorption spectrum of ceruloplasmin is shown in Fig. 2. Two e x t r ema located at 1632 and 1530 cm -1 can be distinguished. Circular dichroism Fig. 3 is a CD difference spectrum of human ceruloplasmin and its apoprotein, showing the cont r i but i on o f the copper chromophores. The spectrum extends from a b o u t 325 to a b o u t 700 nm. Below 325 nm, the c o n t r i b u t i o n of the aromatic amino acid residues to the CD spectra o f apo- and ceruloplasmin is large compared to t he c o n t r i b u t i o n of the c op per chrom ophores, and the determination o f the CD difference becomes uncertain due to the large error involved. Resolution into cons t i t ue nt bands is indicated in Fig. 3. The resolved bands located at 14 000, 16 260, 18 350 and 22 000 cm -l have the same band positions and bandwidths as their counterparts in the resolved absorption spectrum. The residual CD can be fitted with a curve whose shape is the derivative o f t h e Gaussian absorption band located at 31 000 c m - ' . The conditions under which the CD curve may be a p p r o x i m a t e d by t he derivative of a Gaussian have been discussed b y T i noco [13] and Brahms and Brahms [ 1 4 ] . The parameters o f the resolved CD bands are given in Table I. T h e CD spectrum of ceruloplasmin in the ultraviolet range is given in Fig. 4. A t ro u g h at 219 nm is indicated. E m issio n The fluorescence spectra of native and apoceruloplasmin are shown in Fig. 5. The positions o f the maxima are consistent with t r y p t o p h a n fluorescence.

136

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500

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-0.4

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2.0 ~ X 10 -4, cm

1.5

-1

F i g . 3. D i f f e r e n c e C D s p e c t r u m o f h u m a n n a t i v e a n d a p o e e r u l o p l a s m i n . T h e o r d i n a t e r e p r e s e n t s t h e m o l a r circular dichroic absorptivity, calculated on the basis of the total copper content of ceruloplasmin. Observed values (o) are readings at intervals of 5 nm of the recorded spectrum. The solid line ( ) is the calculated sum of constituent bands of Gaussian shape (...... ) and a band (. . . . . ) whose shape is t h e d e r i v a t i v e o f t h e r e s o l v e d a b s o r p t i o n h a n d l o c a t e d a t 3 1 0 0 0 c m -1 . C o n d i t i o n s a s i n F i g . 1.

I

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220

I

240

Ik, n r n Fig. 4. Ultraviolet CD spectrum of human e l l i p t i e i t y . C o n d i t i o n s a s i n F i g . 1.

eeruloplasmin.

The

ordiante

represents

the

mean

residue

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F i g . 5. F l u o r e s c e n c e s p e c t r a o f a p o ( 280 nm. The difference spectrum, obtained acetate, pH 6.0, and 25°C.

) a n d n a t i v e (. . . . . ) h u m a n c e r u l o p l a s m i n , e x c i t e d at b y s u b t r a c t i o n , is a l s o s h o w n ( . . . . . . ). C o n d i t i o n s : 0 . 1 M

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550

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F i g . 6. P h o s p h o r e s c e n c e s p e c t r u m o f h u m a n c e r u l o p l a s m i n , e x c i t e d a t 2 4 0 n m . T h e i n d i v i d u a l c o n t r i b u t i o n s o f t y r o s i n e (. . . . . ) a n d t r y p t o p h a n (...... ) were obtained by resolution of the observed phosphorescence spectrum according to Shaklai and Daniel [12]. Insert: Decay of the phosphorescence e m i s s i o n o b s e r v e d at 3 7 0 (e l ) a n d 4 4 0 (o 0) n m . C o n d i t i o n s : 0 . 5 % g l u c o s e , 0 . 1 M a c e t a t e , pH 6.0, 77°K.

138 TABLE

II

FLUORESCENCE

AND PHOSPHORESCENCE

R e s u l t s axe m e a s u r e d

Fluorescence intensity

Apoceruloplasmin Native ceruloplasmin

PARAMETERS

IN HUMAN

CERULPLASMIN

in 0.1 M acetate, pH 6.0.

1.00 0.69

Phosphorescence

lifetimes * (s)

Tyrosyl

Tryptophanyl

Trp : Tyr phosphorescence ratio **

1.4 1.5

6.0 4.7

11.5 5.3

* Determined from the decay curve of the phosphorescence at 370 and 440 nm for the tyrosyl and and tryptophanyl components, respectively. ** Calculated by comparison of the areas under the resolved bands of the phosphorescence spectra.

Relative to the apoprotein, the fluorescence intensity of the native ceruloplasmin is partially quenched, and the maximum of the emission shifted to the blue. The phosphorescence spectrum of ceruloplasmin is shown in Fig. 6. Resolution of the spectrum in a manner analogous to that used for hemocyanin [12] shows the separate contributions of tyrosine and tryptophan. The lifetimes of the tyrosyl and t r y p t o p h a n y l phosphorescence were determined by following the decay of the emission at 370 and 440 nm, respectively (Fig. 6, insert). Similar measurements were carried out on apoceruloplasmin. It was found that removal of copper, while not affecting the tyrosyl lifetime, brings about an increase in the lifetime of t r y p t o p h a n phosphorescence and a decrease in the tyrosyl to t r y p t o p h a n y l intensity ratio {Table II). Discussion

The absorption spectrum of human ceruloplasmin has been described by Holmberg and Laurell [15], and by Blumberg et al. [16]. The CD spectrum in the visible and near infrared region was reported by Falk and Reinhammar [17], and by Herve et al. [18]. Blumberg et al. [16] resolved the absorption spectrum into four Gaussian bands. The resolution carried out in the present study is based on the simultaneous consideration of both absorption and CD spectra, and indicates the presence of at least six transitions. With the exception of one, the c o m p o n e n t bands have identical locations and bandwidths in absorption and CD (Case I of Moffitt and Moscowitz [19]). To the absorption band centered at 31 000 cm -~ and loosely termed the 330 nm band, there corresponds in the CD spectrum a couplet (Schellman [20]), a curve characterized by both negative and positive ellipticities, with a crossover at the wavelength of the absorption maximum. The large error involved in the determination of the CD difference spectrum at wavelengths less than 325 nm (see Results) precluded measurements in the region of negative ellipticities. Examination of the spectra reported by Falk and Reinhammer [17] and by Herve et al. [18] reveals positive ellipiticties below 400 nm and a crossover approx. 330 nm, and indicates that our experimental findings in this region are reliable. The occurrence of a couplet in CD is a manifestation of exciton splitting and requires interaction between two, or more, similar monomeric chromophores [ 13]. Our analysis of the data indicates that two or more coppers are involved

139 in the chromopore responsible for the 330 nm absorption band. The presence of /3 structure in human ceruloplasmin is indicated by its infrared absorption spectrum. According to Krimm [21] and Miyazawa and Blout [22], strong bands at 1632 and 1530 c m - ' are characteristic of a pleated sheet conformation in polypeptides and proteins. The occurrence in the CD spectrum of a trough at 219 nm, similar to the one observed [23,24] in the form of poly(L-lysine), may also be taken as an indication of the presence of /~ conformation in this protein. The absence of a peak or shoulder at about 206 nm strongly suggests that very few or no amino acid residues have the a-helical conformation and that, as in the case of concanavalin A [25--27], residues not included in the fl structure are in an unordered form. The fractions of amino acid residues in the two conformations may be calculated from the magnitude of the ellipticity, if one assumes the additivity of their optical activity [ 28--30]. Taking the ellipticity values determined by Greenfield and Fasman [28], we estimate that 0.46 of the residues in human ceruloplasmin are in the conformation, the remaining 0.54 being in an unordered form. Lower estimates (0.20 and 0.31) of ~ structure in human ceruloplasmin have been reported by Moshkov et al. [31] and Herve et al. [32]. The presence of fi structure in porcine ceruloplasmin has been reported by Hibino et al. [33]. Comparison of the fluorescence of native ceruloplasmin with that of its apoprotein reveals that the presence of copper brings about a decrease of the t r y p t o p h a n y l fluorescence. The overlap between the fluorescence band (Fig. 5) and the 330 nm absorption c o m p o n e n t band (Fig. 1) suggests that the quenching can be attributed to long-range radiationless energy transfer [34] from the t r y p t o p h a n y l residues acting as donors to the copper chromophores acting as acceptors. It may be pointed o u t that copper has been reported to cause a strong quenching of t r y p t o p h a n fluorescence in Pseudomonas fluorescens azurin, a bacterial protein which contains one copper atom per molecule [35]. In the case of azurin, however, the quenching could not be attributed to long-range energy transfer, since the absorption spectrum of this protein lacks the 330 nm absorption band of the multi-copper proteins. In addition to its quenching effect at the singlet level, copper causes a quenching of t r y p t o p h a n y l emission at the triplet level, as evidenced by the shortened lifetime of t r y p t o p h a n y l phosphorescence in ceruloplasmin relative to its apoprotein (Table II). In contrast, the fact that the phosphorescence lifetime of tyrosine is virtually equal in native and apoceruloplasmin indicates that the copper does not cause quenching of the tyrosine from the triplet. The ratio of the t r y p t o p h a n y l phosphorescence intensities in ceruloplasmin and its apoprotein, determined from the data in Table II, on the assumption that the tyrosyl intensity is unaffected by copper, is 5.3 : 11.5 = 0.46. This ratio is in reasonable agreement with the value of 0.69 (4.7 : 6.0) = 0.54, calculated on the basis of the quenching observed in the fluorescence intensities and the lifetime ratio of the t r y p t o p h a n y l phosphorescence in ceruloplasmin and its apoprotein. The number of coppers per molecule of ceruloplasmin is n o t known with certainty. Recent work [2] indicates that the number is six, and our results suggest that it could be as low as five. Malmstrom, Vanngard and their coworkers [36--38] introduced the now widely accepted classification of coppers in blue proteins according to which three types of copper can be

140

distinguished: type I, associated with 610 nm absorption; type II, not associated with any detectable absorption; and type III, responsible for 330 nm absorption. The interaction demonstrated in the present study between the coppers responsible for the 330 nm band is consistent with the accumulating evidence that this chromophore occurs as an EPR non-detectable spin-paired Cu2+-Cu 2÷ unit [6, 38]. References 1 S c h e i n b e r g , I.H. ( 1 9 6 6 ) in T h e B i o c h e m i s t r y o f C o p p e r ( P e i s a c h , J., Aisen, P. a n d B l u m b e r g , W.E., eds.), p p . 5 1 3 - - 5 2 4 , A c a d e m i c Press. N e w Y o r k 2 M a g d o f f - F a i r c h i l d , B., Lovell, F . M . a n d L o w , B.W. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 3 4 9 7 - - 3 4 9 9 3 R y d e n , L. ( 1 9 7 2 ) E u r . J. B i o c h e m . 26, 3 8 0 - - 3 8 6 4 F r e e m a n , S. a n d D a n i e l . E. ( 1 9 7 3 ) B i o c h e m i s t r y 12, 4 8 0 6 - - 4 6 1 0 5 v a n L e e u w e n , F . X . R . , Wever, R . a n d v a n G e l d e r , B . F . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 5 , 2 0 0 - - 2 0 3 6 C a r r i c o , R . J . , M a l m s t r o m , B.G. a n d V a n n g a r d , T. ( 1 9 7 1 ) E u r . J. B i o c h e m . 2 0 , 5 1 8 - - 5 2 4 7 D e i n u m , J. a n d V a n n g a r d , T. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 0 , 3 2 1 - - 3 3 0 8 K a s p e r , C.B. a n d D e u t s c h , H , F . ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 2 3 2 5 - - 2 3 3 7 9 Daniel, E. a n d Y a n g , J . T . ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 , 5 0 8 - - 5 1 2 1 0 H o l z w a r t h , G. a n d D o t y , P, ( 1 9 6 5 ) J. A m . C h e m . S o c . 8 7 , 2 1 8 - - 2 2 8 11 Miles, D.W. a n d U r r y , D.W. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 2 7 9 1 - - 2 7 9 9 1 2 S h a k l a i , N. a n d D a n i e l , E. ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 2 1 9 9 - - 2 2 0 3 13 T i n o c o , I. ( 1 9 6 8 ) J. C h i m . P h y s . 6 5 , 9 1 - - 9 7 1 4 B r a h m s , J. a n d B r a h m s , S. ( 1 9 7 0 ) in F i n e S t r u c t u r e of P r o t e i n s a n d N u c l e i c A c i d s ( F a s m a n , G . D . a n d T i m a s h e f f , S.N., eds.), p. 2 1 4 , M a r c e l D e k k e r , N e w Y o r k 1 5 H o l m b e r g , C . G . a n d L a u r e l l , C.-B. ( 1 9 4 8 ) A c t a C h e m . S c a n d . 2, 5 5 0 - - 5 5 6 16 B l u m b e r g , W.E., E i s i n g e r , J., Aisen, P., Morell, A . G . a n d S c h e i n b e r g , I.H. ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 1675--1682 17 F a l k , K.-E. a n d R e i n h a m m a r , B. ( 1 9 7 2 ) B i o c h i m , B i o p h y s , A c t a 2 8 5 , 8 4 - - 9 0 1 8 H e r v e , M., G a r n i e r , A., Tosi, L. a n d S t e i n b u c h , M. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 9 , 4 3 2 - - 4 4 1 1 9 M o f f i t t , W. a n d M o s c o w i t z , A. ( 1 9 5 9 ) J. C h e m . P h y s . 3 0 , 6 4 8 - - 6 6 0 2 0 S c h e l l m a n , J . A . ( 1 9 6 8 ) A c c . C h e m . Res. 1, 1 4 4 - - 1 5 1 21 K r i m m , S. ( 1 9 6 2 ) J. Mol. Biol. 4, 5 2 8 - - 5 4 0 2 2 M i y a z a w a , T. a n d B l o u t , E . R . ( 1 9 6 1 ) J. A m . C h e m . Soc. 8 3 , 7 1 2 - - 7 1 9 23 S a r k a r , P . K . a n d D o t y , P. ( 1 9 6 6 ) P r o c . N a t l . A c a d . Sci. U.S. 55, 9 8 1 - - 9 8 9 24 T o w n e n d , R., K u m o s i n s k y , T . F . , T i m a s h e f f , S.N., F a s m a n , G . D . a n d D a v i d s o n , B. ( 1 9 6 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 2 3 , 1 6 3 - - 1 6 9 2 5 K a y , C.M. ( 1 9 7 0 ) F E B S L e t t . 9, 7 6 - - 8 0 26 E d e l m a n , G . M . , C u n n i n g h a m , B.A., R e e k e , G . N . , B e c k e r , J.W., W a x d a l , M.J. a n d W a n g , J . L . ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U.S. 6 9 , 2 5 8 0 - - 2 5 8 4 27 H a r d m a n , K . D . a n d A i n s w o r t h , C . F . ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 4 9 1 0 - - 4 9 1 9 2 8 G r e e n f i e l d , N. a n d F a s m a n , G . D . ( 1 9 6 9 ) B i o c h e m i s t r y 8, 4 1 0 8 - - 4 1 1 6 2 9 S a x e n a , V.P. a n d W e t l a u f e r , D.B. ( 1 9 7 1 ) P r o e . N a t l . A c a d . Sci. U.S. 6 8 , 9 6 9 - - 9 7 2 3 0 C h e n , Y . H . , Y a n g , J . T . a n d M a r t i n e z , H . M . ( 1 9 7 2 ) B i o c h e m i s t r y 11, 4 1 2 0 - - 4 1 3 1 31 M o s h k o v , K . A . , Vasilets, I.M. a n d K u s h n e r , V . P . ( 1 9 7 2 ) Mol. Biol. 6, 4 1 - - 4 8 3 2 H e r v e , M., G a r n i e r , A., T o s i , L. a n d S t e i n b u c h , M. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 0 5 , 3 1 8 - - 3 2 3 3 3 H i b i n o , Y., S a m e j i m a , T., K a j i y a m a , S. a n d N o s c h , Y. ( 1 9 6 9 ) A r c h . B i o c h e m . B i o p h y s . 1 3 0 , 6 1 7 - - 6 2 3 3 4 F o r s t e r , T. ( 1 9 5 9 ) Disc. F a r a d a y S o c . 27, 7 - - 1 7 3 5 F i n a z z i - A g r 6 , A., R o t f l i o , G., A v i g l i a n o , L., G u e r r i e r i , P., Boffi, V. a n d M o n d o v i , B. ( 1 9 7 0 ) B i o c h e m i s t r y 9, 2 0 0 9 - - 2 0 1 4 3 6 M a l k i n , R, a n d M a l m s t r o m , B . G . ( 1 9 7 0 ) A d v . E n z y m o l . 3 3 , 1 7 7 - - 2 4 4 3 7 V a n n g a r d , T. ( 1 9 7 2 ) in B i o l o g i c a l A p p l i c a t i o n s o f E l e c t r o n S p i n R e s o n a n c e ( S w a r t z , H . M . , B o R o n , J . R . a n d B o r g , D.C., eds.), p. 4 1 1 , J o h n Wiley a n d S o n s , I n c . , N e w Y o r k 3 8 R. M a l k i n ( 1 9 7 3 ) in I n o r g a n i c B i o c h e m i s t r y ( E i c h o r n , G . L . , e d . ) , V o l . 2, p p . 6 8 9 - - 7 0 9 , Elsevier Sci. P u b l . Co., N e w Y o r k 3 9 M u l l i k e n , R . S . ( 1 9 3 9 ) J. C h e m . P h y s . 7, 1 4 - - 2 0