238

Biochimica et Biophysica Acta, 537 ( 1 9 7 8 ) 2 3 8 - - 2 4 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38035

E L E C T R O N N U C L E A R DOUBLE RESONANCE OF CYTOCHROME OXIDASE: N I T R O G E N AND P R O T O N E N D O R FROM THE 'COPPER' EPR SIGNAL *

H.L. V A N C A M P a , * * , Y.H. WEI b, C.P. S C H O L E S a a n d T S O O E. K I N G b

a Department o f Physics and Center for Biological Macromolecules, and b Laboratory o f Bioenergetics, State University o f New York at Albany, Albany, N.Y. 12222 (U.S.A.) ( R e c e i v e d April 2 0 t h , 1 9 7 8 )

Summary Proton E N D O R resonances have been found from at least two different protons with fairly large and isotropic couplings of a b o u t 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H20. We have obtained nitrogen E N D O R from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have n o t y e t demonstrated experimentally E N D O R characteristic of the copper nucleus itself.

Introduction C y t o c h r o m e oxidase is the terminal enzyme in the intracellular respiration of obligatory aerobes. The functional unit of the oxidase of mammals, yeast and certain other phyla most probably contains two hemes (heme a) and t w o coppers [1], and its monomeric molecular weight has been estimated in the neighborhood of 100 000 [2]. The integrated EPR intensity of the fully oxidized oxidase can account for less than half of the paramagnetic centers chemically determined [3,4], and it has been suggested that one heme and one copper are probably antiferromagnetically coupled [5,6]. Of the EPR signals that are seen in the fully oxidized oxidase, one is a low spin ferric, cytochrome-like signal, and the other is the so-called 'intrinsic copper' signal near g = 2.0 with* A part of the material containe~l in this p a p e r w a s p r e s e n t e d at t h e March 1978 Biophysical Society Meeting, Washington, D.C. (Abstract No. M-PM-A1). ** P r e s e n t address: B i o p h y s i c s R e s e a r c h Laboratory, University of Michigan, Ann Arbor, Mich. 48104, U.S.A.

239 out hyperfine structure (although recent computer simulations have suggested small copper hyperfine couplings in the 20--33 gauss range; i.e., 48 to 80 MHz range [7]). In contrast, upon denaturation the intrinsic signal disappears and a typical inorganic copper signal appears in its place [8]. The intrinsic 'copper' has a gll = 2.18 and g l ~ 2.00; recent Q-band studies [4] and computer simulations [4,7] show a rhombic splitting of g± into gx = 1.99 and gy = 2.03. The g-values (particularly gx and gy) are quite low for copper complexes. Unusually small copper hyperfine couplings and g-values have been attributed in copper proteins to tetrahedral perturbations which mix copper 4s and 4p orbitals into copper 3d [7,9]. Sulfur and nitrogen ligands are most frequently mentioned as ligands for copper in proteins [10,11]. We believed that any hyperfine information on the nuclei coupled to the paramagnetic electron of the 'copper' signal would be most useful, and electron nuclear double resonance (ENDOR) is an effective tool for resolving hyperfine couplings. A variety of low molecular weight copper complexes have already been studied by ENDOR when substituted into diamagnetic hosts [12--15]. Nitrogen hyperfine couplings, nitrogen quadrupole interactions, and proton hyperfine couplings have been seen. In two instances [14,15] ENDOR of the copper nucleus itself was seen in the 40--50 MHz region. ENDOR has also been done on the copper-containing protein stellacyanin, and protons and somewhat unresolved nitrogens were seen [16]. The ENDOR technique has been used to obtain hyperfine couplings to atoms of heme proteins [17,18] and hemins [19,20]. Many previous ENDOR studies were from frozen solutions or powders. If g axes and hyperfine axes are co-linear, ENDOR spectra with resolution comparable to spectra from single crystals can be obtained from non-crystalline material by sitting at the g-value extrema. For this initial study we have concentrated much of our ENDOR effort on the g-value extremes of the 'copper' EPR signal. A practical reason for looking first at ENDOR from the 'copper' signal is that in the oxidase this signal has much greater intensity than heme a because the 'copper' signal is spread over only a few hundred gauss whereas the low spin ferric heme signal is spread over several thousand gauss. Experimental The ENDOR apparatus used for these studies is described in detail in Refs. 18 and 19. For assignment of ENDOR signals we used several different plastic [21] EPR-ENDOR cavities which resonated in the 9.00 to 9.39 GHz range. We did our ENDOR work at 2.1°K with microwave powers of about 10/~W and the dispersion (X') m o d e under rapid passage conditions. ENDOR radio frequency was typically 1/2 gauss peak to peak. For ENDOR of the more strongly coupled protons and for the nitrogens we used 100 kHz field modulation amplitudes of about 6 gauss peak to peak and an ENDOR frequency sweep rate of about 6 MHz/s. Better resolution of more weakly coupled protons was obtained with a small 100 kHz field modulation of about 0.3 gauss peak to peak and a slower radio frequency sweep rate of about 0.1 MHz/s. Cytochrome oxidase was isolated and purified by previously published methods [22,23]. Samples prepared by this m e t h o d typically contain 25--50

240 mg protein/ml and 10 nmol heme a/mg protein, and they have a copper: heme ratio n o t higher than 1.1. Enzymic activity was determined from 02 consumption in the presence of reduced c y t o c h r o m e c and ascorbate at about 22°C. As previously described, the activity of purified enzyme can be increased by adding lipid to purified enzyme. The 02 turnover decreases from a b o u t 10 nmol 0 2 " s -1" nmo1-1 heme a in the presence of phospholipid to two orders less activity in its absence [22]. The E N D O R spectra have so far shown no dependence on phospholipid-induced activity changes. To check for E N D O R of exchangeable protons, several samples of purified oxidase were suspended in and dialyzed against 99% deuterated phosphate buffer that had 1% sodium cholate added. Samples of a b o u t 1.5 ml volume were prepared in 0.05 M potassium phosphate buffer (pH 7.4), 1% cholate, and then the samples p u t into 7 mm (internal diameter) tubes and frozen by plunging into liquid nitrogen. The oxidase samples will often contain a very small amount of adventitious copper. The a m o u n t of adventitious copper will increase to a certain extent with preparative manipulation. Thus several samples were quickly prepared w i t h o u t going through all the steps for native lipid removal [22]. Also buffer containing 25 mM EDTA (suggested by C. Hartzell) was used in several preparations as a precaution against adventitious copper. E N D O R frequencies and E N D O R intensity (as a percentage of EPR signal) showed no correlation with adventitious copper *

EPR and ENDOR results Fig. 1 shows the EPR spectra of c y t o c h r o m e oxidase taken at 80 K and at 4.2 K. The EPR spectrum at 4.2 K is similar to that at 80 K except that the EPR lines are narrower and at 4.2 K a small peak from ferric heme a, labeled a in Fig. 1, appears near g = 2.25. (Compare Fig. 2A of ref. 4.) The positions A (g = 2.00) and B (g = 2.18) were the positions where the E N D O R spectra shown here were taken. E N D O R spectra were seen in the 1--30 MHz region, and these spectra, as shown in Fig. 2, are n o t at all like those seen from ferric heme proteins [18]. The major features of the spectra in Fig. 2 are quite similar at positions A and B, and indeed they stay similar at intervening g-values. The two large peaks in the 18--24 MHz region, which we have shown to be protons, each did show a small splitting of a b o u t 1 MHz at g-values around 2.1. Such a splitting may suggest that each peak has contributions from more than one proton or suggest a small anisotropy in proton hyperfine couplings that is not obvious at positions A or B. To assign the major features of our E N D O R signal, we sat at position A (where EPR and E N D O R signals are largest) with two different EPR-ENDOR cavities that resonated at 9.00 and 9.39 GHz. As shown in Fig. 3, the E N D O R lines in the 7--10 MHz region did n o t move (in accord with their being from * O n e of o u r o x i d a s e s a m p l e s ( n o t c o n t a i n i n g E D T A ) w a s i n a d v e r t e n t l y f r o z e n and s l o w l y t h a w e d in prep a r a t i o n , This s a m p l e s h o w e d a large a d v e n t i t i o u s i n o r g a n i c c o p p e r EPR signal w i t h characteristic wellr e s o l v e d n i t r o g e n h y p e r f i n e splittings ( ~ 1 5 gauss) in the g± r e g i o n [ 7 ] . T h i s s a m p l e s h o w e d a severalM H z - b r o a d E N D O R signal, w h i c h w a s c e n t e r e d at a b o u t 1 7 M H z and w h i c h w a s n o t s e e n in b e t t e r o x i d a s e s a m p l e s . T h i s s a m p l e also s h o w e d t h e u s u a l E N D O R p e a k s in t h e 7 - - 1 0 M H z and 1 8 - - 2 4 M H z region, albeit somewhat more weakly.

241

CYTOCHROMEc OXIDASE"(X)PPER"SIGNAL ~gVALUE 2P,O 2.32 2.24 2.16 2.08 200 I

r

i

i

I

I

I

I

I

I

I

I

~

I

/~Ye =93.5GHz T : 80K

I

I

2800

I

I

I

3000 3200 3400 MAGNETICFIELD(GAUSS)~

3600

Z e.D

~g'VALUE 2.40 2.32 2.24 2.16 2.08

2.00

l.lJ

'

' ~ '

'

' ~'

- - 0

'

'

'~'

/ ~~e=9'.17GHz '

I - ~ t~.z~

\

T :42K

2600 2800 3000 3200 3400 I

I

I

I

MAGNETICFIELD(GAUSS)

Fig. I . E P R traces o f t h e ' c o p p e r ' signal f r o m o x i d a s e . T r a c e s w e r e t a k e n w i t h a f i e l d s w e e p o f I 0 0 0 g a u s s i n 5 m i n . T i m e c o n s t a n t = 0 . 1 s., 1 0 0 K H z m o d u l a t i o n a m p l i t u d e = 6 g a u s s p o i n t t o p o i n t . T h e m i c r o w a v e p o w e r at S 0 K w a s 1 . 3 m M a n d at 4 . 2 K w a s 1.3 /~W. P o i n t s A a n d B s h o w w h e r e E N D O R t r a c e s b e l o w w e r e t a k e n . T h e p e a k l a b e l e d a, w h i c h is r e s o l v e d at 4 K b u t n o t at 8 0 K , is n e a r t h e i n t e r m e d i a t e g-value ( g y ) o f l o w spin c y t o c h r o m e a. T h e s a m p l e u s e d h e r e c o n t a i n e d a b o u t 2 5 m g / m l o f p r o t e i n , 1 0 n m o l h e m e a/mg p r o t e i n a n d h a d a n 0 2 t u r n o v e r rate o f a b o u t 2 n m o l 0 2 • s - I • n m o l - I h e m e a.

'4N), while the m o v e m e n t of the lines in the 18--24 MHz region was highly in accord with their being protons. The ENDOR frequencies of the observed protons and nitrogens are given in Table I. Fig. 4 shows weakly coupled protons near the free proton frequency. There is a substantial difference in proton spectra at positions A and B. It is possible that at position B the ferric cytochrome in the oxidase may make a small contribution to the proton ENDOR in Fig. 4. Nearby at g = 2.25 on the cytochrome signal there is a poorly resolved, weakly coupled proton ENDOR signal of about 1/5 the intensity of the spectrum of Fig. 4B. We have in addition looked for E N D O R characteristic of the copper nuclei, 63Cu and 6SCu, and have seen at best a barely resolved broad hump in the 30--40 MHz region at g = 2.18, which we do n o t at all consider as definite ENDOR evidence for copper.

242 STRONGLY COUPLED NUCLEI I

I

POSITION

I

I

I

I

A

I I

I

I

I

21K t/e : 914GHz H = 527kG T :

t v

POSITION g //e : 914 GHz

H : 2 99kG %=218

I 4.0

1 70

I I LO.O I$.0

t t 16.0 190 220

t ~ 250 280

FREQUENCY [ M H z ] ~

Fig. 2. E N D O R t r a c e s t a k e n t o s h o w s t r o n g l y c o u p l e d p r o t o n s a n d n i t r o g e n s at p o s i t i o n s A ( n e a r g.L) a n d B ( n e a r gll)" T r a c e s w e r e t a k e n u n d e r c o n d i t i o n s d e s c r i b e d as a p p r o p r i a t e f o r n i t r o g e n a n d s t r o n g l y c o u p l e d p r o t o n s in t h e e x p e r i m e n t a l section. T h e r e s p e c t i v e free p r o t o n f r e q u e n c i e s (UNMR) ate i n d i c a t e d o n e a c h figure. E N D O R t r a c e s w e r e d o n e w i t h a m i c r o w a v e c a v i t y w h i c h r e s o n a t e d at Ue = 9 . 1 4 G H z . S a m p l e t h e s a m e as f o r Fig. 1.

ASSIGNENDORin 7-I0 MHz REGION ~o L4N POSITIONA

Lw ' RROFFREEPROTONS

I

- [368 MHz -

I

I

i

[

I

I

/~

I

I

I

/

I

,

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I I

i] I

i I

ASSIGN ENDORin 18-24 MHz REGION Io PROTONS I I I I I ~, I I-~

I T :21K

I

PORTION ~ t

11e :9 O0 GH

~'~/

H =32~kG I\

I

i\

(Je: 2 O0

;

'

.... ()F FREE PROTO'NS _1368 MHZl I I ~

,

,

'\ ,

II

I

>

/

,

I ~'. ,

! 21K

T

ge: ~00

z:

z ~o

POSITIONA l I I 1 I I

c] z L~J

I /

I I

YN~OFFREEPROTONS - 1427 MHz~ I i I I I J ~ j i I / i\ I I I I /

I "\-'~h J// ~2gN¢nh =206~/~'~Z~

60

l

I

I

70

80

90

i00

I k

I

I

L i I

T=21K I~e : 939 GHz H : 335kG ge: 2 O0

I

ROS]TION

A

'i

/~ I

,'

L.~

I I

~

I I

J

I

q

i

I

I

--

I

I

I

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~

,I" ~ ge '200H: 3,SkG " I

Ue 9 $9 GFtz

I L\ I

I o ]

I I 110

180 120

I v~M~OFFREEPROTONS

1

I

~

I

L~

I

190

200

-1427MHz-

I

I

21.0 220

"

I 250

240

250

260

FREQUENCY [MH z ]

FREQUENCY[MHz]

Fig. 3. E N D O R t r a c e s t a k e n a t g = 2 . 0 0 f o r t h e p u r p o s e of assigning t h e m a j o r f e a t u r e s in t h e 7 - - 1 0 M H z r e g i o n a n d t h e 1 8 - - 2 4 M H z r e g i o n . E N D O t t w a s d o n e w i t h cavities w h i c h r e s o n a t e d at 9 . 0 0 a n d 9 . 3 9 G H z , t h u s r e s u l t i n g in t h e d i f f e r e n t r e s p e c t i v e r e s o n a n c e m a g n e t i c fields a n d free p r o t o n f r e q u e n c i e s indic a t e d . E x p e r i m e n t a l c o n d i t i o n s aze" as d e s c r i b e d as a p p r o p r i a t e f o r n i t r o g e n a n d s t r o n g l y c o u p l e d p r o t o n s in t h e e x p e r i m e n t a l s e c t i o n . T h e v a l u e s o f t h e 1 4 N Z e e m a n splitting ( 2 g N ~ n H ) are i n d i c a t e d f o r t h e resp e c t i v e fields b e l o w t h e p e a k s in t h e 7 - - 1 0 M H z r e g i o n . M o v e m e n t of r e s o n a n c e s in t h e 1 8 - - 2 4 M H z r e g i o n i n d i c a t e s p r o t o n s , S a m p l e t h e s a m e as f o r Fig. 1.

243 TABLE I ENDOR FREQUENCIES g-value

H (kgauss)

Nitrogen frequencies 2.18 2.99 2.00

3.27

g-value

H (kgauss)

Proton frequencies 2.18 2.99

ENDOR Frequency (MHz)

2gN~n H ( M H z )

7.67 9.41 7.80 9.82

1.84

+ 0.10 ± 0.10 ± 0.10 -+ 0 . 1 0

2.01

VNM R ( M H z )

ENDOR Frequency (MHz)

12.73

Strong couplings 18.72 ± 0.10 22.29 ± 0.15

A (MHz)

11.98 ± 0.20 19.12 + 0.30

Weak couplings

2.00

3.27

13.90

a, 6'

12.10 ± 0.09 13.43 ± 0.09

1 . 3 3 _+0 . 1 3

~, ~'

12.59 ± 0.03 1 3 . 0 0 _+0 . 0 3

0.41 ± 0.04

Strong couplings 19.85 ± 0.15 23,36 + 0.15

11.90 ± 0.30 18,92 ± 0.30

Weak eouplings D, D'

12.84 + 0.09 } 15.12 + 0.09

2.28 + 0.13

C, C'

1 3 . 3 3 -+ 0 . 0 3 } 14.56 ± 0.03

1.23 + 0.04

B, B '

13.55 ± 0.03 1 4 . 3 5 -+ 0 . 0 3 ~

A, A '

13.80 ± 0.03 ~ 1 4 . 1 0 + 0.03

0.80 ± 0.04 0.30 ± 0.04

* N i t r o g e n a n d s t r o n g l y c o u p l e d p r o t o n f r e q u e n c i e s w e r e o b t a i n e d f r o m t h e a v e r a g e o f a l t e r n a t e l y inc r e a s i n g a n d d e c r e a s i n g f r e q u e n c y s w e e p s . W e a k l y c o u p l e d p r o t o n f r e q u e n c i e s w e r e o b t a i n e d f r o m increasing frequency sweeps.

Theory and Discussion We interpret our E N D O R data with a first order spin Hamiltonian [18--20]. For ~4N one expects to see E N D O R transitions occurring in pairs as follows: /)ENDOR = A / 2 + P + glv[JnH ;

A /2 - - P + gNfl, H

(1)

where A is the hyperfine coupling along the direction of the applied field, gN is the ~4N nuclear g-value, gN~,,H is the nuclear Zeeman interaction (~ 1 mHz

244 WEAKLY COUPLED PROTONS I

I----AI

POSITION A

I

B

I T : 2 1K

B'

~e : 914 GHz H : 327 kG

C'

5~

1 "-I-

/

~ ILO

ge : 2 O0

\o,

T

~N~ROF FREE PROTONS~

I

I

I

120

130

140

.~.~._

_1

I 160

150

170

Z ,.y0 ¢'m Z I.i.J

I

I

I

A ,~ /

I I00

I I10

I 120

I ~,

cf

POSITION B

......

i

/"X

I

T :24K

/ \ ~'

~e:gl4GHz

/

H: 2 99 kG

'.#x_~

L . . . . . . . . . . 1__ 130 140

I

t50

FREQUENCY [MHz] Fig. 4. ENDOR

at p o s i t i o n s A a n d B f r o m w e a k l y c o u p l e d p r o t o n s n e a t t h e t e e p r o t o n f r e q u e n c y . T r a c e s w e r e t a k e n u n d e r c o n d i t i o n s d e s c r i b e d a s a p p r o p r i a t e f o r w e a k l y c o u p l e d p r o t o n s in t h e e x p e r i m e n t a l section. Peaks (or shoulders) labeled with the same letter ate from the same proton. ENDOR traces were d o n e w i t h a m i c r o w a v e c a v i t y w h i c h r e s o n a t e d at v e = 9 . 1 4 G H z . S a m p l e t h e s a m e f o r F i g . 1.

here), and P is the c o m p o n e n t of the quadrupole interaction along the direction of the applied field. The Zeeman splitting of the pairs of lines is 2gN[J,,H. The splitting of the lines which we saw in the 7--10 MHz region was very close to that expected for 14N, as indicated in Fig. 3. We have wondered where the other t w o expected nitrogen ENDOR lines may be. It is possible that there are only two nitrogen lines because P ~ O or else that the expected other two lines are obscured near the free proton frequency. (Interference between protons and other nuclei near the free proton frequency has previously been seen by ENDOR [ 1 8 , 1 9 ] . ) The ENDOR frequencies of the two observed nitrogen lines remain very isotropic at all electronic g-values between 2.18 and 2.00. The average frequency of coupling to the observed nitrogens corresponds to an isotropic coupling of about 17 MHz (6 gauss), which is a reasonable nitrogen-tocopper coupling [24]. Even if we have not detected the possibly obscured partners of the nitrogens in the 7--10 MHz region, the nitrogen couplings will still be in the 10--25 MHz region because quadrupolar splittings for nitrogen are only a few MHz [12,13]. For protons the first-order expression for ENDOR frequencies is: b ' E N D O R ---- /2NM R +- A/2 (2) Proton ENDOR lines normally appear in pairs centered about the free proton Zeeman frequency, /2NMR, "of order 14 MHz here. Such pairs are definitely seen for the weakly coupled protons in Fig. 4. However, if the proton hyperfine coupling A is comparable to VNMR, it can happen that the ENDOR transition

245 probabilities for the two E N D O R lines above and below VNMRm a y markedly differ [25]. Thus we must experimentally resort to another method to assign the observed E N D O R lines in the 18--24 MHz region. In the experiments done with cavities which resonate at 9.00 and 9.39 GHz, where one sits at g = 2.00, one has a difference in magnetic field of a b o u t 140 gauss and thus expects movement of about 0.6 MHz for protons, b u t considerably less for other conceivable nuclei, notably 14N, ~aCu, or ~SCu *. (For example, the corresponding movement of copper ENDOR lines would be a b o u t 0.15 MHz.) The movement of a b o u t 0.60 MHz was indeed seen for the E N D O R lines in the 18--24 MHz region, as shown in the right half of Fig. 3. Thus we assign these lines as protons. The coupling to these protons is fairly isotropic with values of 12 and 19 MHz. One would expect such large, isotropic couplings from protons of CH or CH2 groups which are one covalent bond removed from the point of maximum electron spin density [26]. Conceivably CH2 protons near a sulfur ligated to copper would be candidates for the observed protons. The weakly coupled protons near the free proton frequency have quite anisotropic couplings. It is very likely that the coupling to these protons is largely dipolar. (A proton 5 -~ from a spin 1/2 center experiences a dipolar interaction varying in magnitude between 1.2 and 0.0 MHz, depending on the angle between the applied magnetic field and the vector from the proton to the paramagnetic center [17]). When we compared the E N D O R signals from protein which had been deuterated with ENDOR of non-deuterated protein, no significant changes were seen in the ENDOR of either the weakly or strongly coupled protons. This implies that the observed protons are n o t exchangeable, at least u n d e r t h e conditions we used. One expects three pairs of ENDOR transitions from I = 3/2 nuclei like 63Cu or 6SCu: VENDOR = A / 2 +: gcufl,,H ;

A / 2 +- gcut3,,H + 2 P ;

A/2 + gcul3,H -- 2P

(3)

One would expect pairs of lines split by the appropriate value of 2gcufl,,H for the copper nuclei, which is in the 7--8 MHz range. We have n o t seen such splitting and have n o t assigned any of the observed E N D O R to copper. It may be that we have simply not found the right temperature and relaxation conditions for copper ENDOR [27]. Possibly g-axes and copper hyperfine and quadrupole axes are non-colinear; such non-coincidence would broaden the E N D O R lines even at g-value extrema [16]. It has been suggested that copper is really not part of the EPR-visible 'copper' center in oxidase; a sulfur radical might be involved [10]. (However, one would n o t expect a nitrogen, such as we already have seen by ENDOR, to be strongly coupled to a sulfur radical.) A detailed E N D O R study of proton and nitrogen couplings is planned at closely spaced points across the EPR line in hope of constructing a detailed model of the vicinity of the paramagnetic center. Measurements of the electron spin-lattice relaxation rate are planned to find better relaxation conditions for doing ENDOR, to obtain more information on the electronic states of the 'cop* This is b e c a u s e the nuclear g-values for 1 H, 14N, 63Cu and 65Cu are, respectively, 5.586, 0.404, 1.484, 1.590,

246

per', and possibly to obtain information on cross-relaxation with other paramagnetic centers within the oxidase. Acknowledgments This work was supported by NIH Grants No. AM-17884, GM-16767 and HL12576. We are grateful to Dr. P.H. Davis for running a number of spectra and for helpful discussion on copper chemistry. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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