Electron-nuclear double resonance of copper complexes of human transferrin

Bwchtmwa et Blophystca Acta, 747 (1983) 49-54

49

Elsewer BBA 31681

ELECTRON-NUCLEAR DOUBLE RESONANCE OF COPPER COMPLEXES OF HUMAN TRANSFERRIN JAMES E ROBERTS a,, THEODORE G BROWN a BRIAN M HOFFMAN a and PHILIP AISEN b **

a Department of Chemtstry, Northwestern Umverslty, Evanston, IL 60201, and bDepartments of Phystology and Blophystcs, and Medwme, Albert Einstein College of Medwme of Yeshwa Umverslty, Bronx, N Y 10461 (U S A ) (Recewed February 21st, 1983)

Key words ENDOR, Copper complex, Transferrm, (Human serum)

Electron-nuclear double resonance (ENDOR) spectroscopy has been used to study ligand and copper hypedine interactions in Cu(lI) complexes of human transferrin. A nearly isotropic superhypedine interaction of the Cu(II) spin with a single 14N nucleus was identified, and the principal values of its tensor were estimated. All principal values of the copper hyperfine tensor were also directly measured for the first time. Resonances from at least two exchangeable protons were observed, but their origin could not be ascertained. At physiological pH, and in the presence of bicarbonate, ENDOR spectra of the two metal-binding sites were virtually indistinguishable.

Introduction

Experimental

The abihty of the two specific metal-binding sites of transferrin to accommodate a wide variety of spectroscopically active metal ions has made this protein a subject of numerous and diverse spectroscopic investigations [1-10]. In particular, continuous-wave and pulsed EPR spectroscopy have been used to analyze the types, numbers and arrangements of hgands at sites beanng Cu(II) [6-10] We now report studies in wluch the enhanced resolution of electron-nuclear double resonance (ENDOR) spectroscopy is exploited to confirm and extend earlier observations by EPR spectroscopy on the copper complexes of human serum transfernn [7-10].

Transferrm complexes Samples for EPR and ENDOR spectroscopy were prepared in 0.1 M KC1/0.05 M Hepes/0.01 M bicarbonate buffer, pH 7.5, as previously described [7,10]. Preparations of four different isotopic ennchments were studied: 65Cu2-transferrin12 ( C O 3 ) 2, 65Cu2-transferrin-Q2CO3)2 exchanged with 2 H 2 0 , 65Cu2-transferrin-(13CO3)2, and 65Cu-transferrin-(12CO3) with copper predormnantly occupying the A-site in the C-tern'anal half of the protein. Samples contaanlng 13C-enriched HCO~- were sealed under vacuum to prevent exchange with atmospheric CO 2.

* Present address Francis B~tter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, U S A ** To whom correspondence should be addressed Abbrevlauon Hepes, 4-(2-hydroxyethyl)-l-plperazaneethanesulfomc aod 0167-4838/83/$03 00 © 1983 Elsevier Sctence Pubhshers B V

EPR and ENDOR spectra EPR and ENDOR spectra were recorded with the spectrometers previously described [ 1l - 13]. ENDOR spectra were obtmned at 2 K in the dispersion mode. Typical conditions were: nucrowave power, 20 pW; field modulation amphtude, 3 G at 100 KHz; and radio frequency amplitude, 1

50 G in the rotating frame. All E N D O R spectra were obtained both with increasing and decreasing radlofrequency sweeps Frequency values given in the text refer to average peak posmons, small shafts from these values, not exceeding 0.3 MHz, are observable in the figures. E N D O R is performed at fixed applied field, H 0, and is manifested by changes in the EPR signal intensity that result from nuclear trans~tlons induced by a swept radlofrequency field Therefore, an E N D O R signal arises from that subset of molecules with orientations such that EPR occurs at fields very near H 0. For Cu(II), the electronic g-tensor typically has axial or nearly axial symmetry, with gll > g±" In thas case, for H 0 set to the low-field edge of the spectrum (field marked A in Fig 1), only those copper centers with gll oriented along H 0 contnbute to the E N D O R signal [14-16]. With H 0 set to the high-field edge, position C, all molecules lying such that H 0 is in the basal plane of the g-tensor can contribute, provided that the in-plane g-anlsotropy is small, as is true in Cu(II)transferrln complexes [6,7] The E N D O R spectrum of a given nucleus will then exhibit maxima at frequencies corresponding to the in-plane extrema of the hyperfine tensor. The normal E N D O R pattern for a set of magnetically equivalent protons is a pair of lines centered at the free proton Larmor frequency, 13.62 M H z at 3200 G [17,18], and separated by the orientation-dependent superhyperfine coupling constants, A r~, with frequencies gwen by Eqn. 1: ~ = I.H + A"/21

(l)

A resonance may be positively assigned to protons by exploiting the linear dependence of n H, the center of the E N D O R pattern, on H 0 and hence the microwave frequency A nucleus, K, having spin greater than one-half (I>~ 1), such as 14N, 63Cu, can exhibit an orientation-dependent quadrupole interaction term, pK, as well as the nuclear Zeeman and hyperflne terms, and its possible E N D O R frequencies are [ 17,18]:

VKm= IAK/2+ v k +(2m - I)pK[

(2)

where - I K + 1 ~< m ~< I K. The result is a pattern having as many as 2 I K lines and centered at the

frequency, AK/2, which, to first order, is independent of the spectrometer microwave frequency. For K = l a N , t, N = 0 984 M H z at 3200 G and a four-hne nitrogen pattern centered at AN~2 is expected. However, in biological systems having lnudazole nitrogens as ligands the quadrupole interaction typically is unresolvably small (Refs. 16, 19-21; and J E. Roberts and B M. Hoffman, unpublished observations), and the pattern reduces to a Larmor-spht doublet centered at AN~2, with a splitting of 2v N. Copper has two isotopes, 63Cu and 6~Cu, both having I = 3 / 2 and Vcu --- 3 8 MHz at 3200 G, and so can exhibit an E N D O R pattern centered at A c u / 2 and having as many as six distinguishable lines. Results and Discussion

EPR Fig. 1 shows X-band (9 21 G H z ) EPR spectra of 65Cu2-transferrln-(12CO3)2 in H 2 0 and 2H20, with characteristics nearly identical to those previously reported [6,7]. Each 65Cu hyperfine line shows a triplet superhyperfine splitting, most clearly seen in the lowest-field (m cu = - 3 / 2 ) line of the sample I n 2 H 2 0 , this splitting (9.0 G; 31 5 MHz) arises from the interaction with a strongly coupled 14N-containing hgand [6]. The gperpendicular regions show a superpositlon of lines

a

.

~,.

:~

11

b A

i/iS1 / ic

Fig 1 EPR spectra of Cu2-transfernn-(t2CO3)2 in (a) H20 and (b) 2H20 T, 77 K, nucrowave frequency, 9 210 GHz F~elds referred to m the &scuss~onof ENDOR are marked A, B, and C

51 TABLE I SPIN-HAMILTONIAN PARAMETERS FOR 65Cu-SUBSTITUTED TRANSFERRIN Measurements on 65Cu-substltuted transfernn m the A-site only, except as noted m the text Tensor dlrecUons are referred to the g-tensor aras g values are from Ref 6 Tensor directions

x g values 14N hyperfine sphttlng (MHz)" pII4N (MHz) b Exchangeable 1H hyperfine sphttmg (MHz) ~ 65Cu hyperfme sphttlng (MHz)

y 2 042 31 5 <03 142 176

z 2 059 31 5 ~<03 94 143

~<88

2312 30 8 <04

g 88

~<502 ~

a EsUmated error ± 1 MHz b Obtained from ]4N ENDOR hnewldth Esumated error ±0 3 MHz c Obtained from EPR (Ref 6) Estimated error ±3 MHz

that p r e s u m a b l y results from a c o m p l e x overlapp i n g of 65Cu a n d 14N sphttings, c o m p o u n d e d b y a small i n - p l a n e g - a n i s o t r o p y . This region has n o t prevaously been a n a l y z e d satisfactorily, so neither the p e r p e n d i c u l a r 65Cu n o r 14N h y p e r f i n e coup h n g c o n s t a n t s are a c c u r a t e l y k n o w n form EPR. P r o t o n h y p e r f i n e sphttings are n o t resolved m the E P R signal, b u t the signal sharpens in 2 H 2 0 , (Fig. 1b), i n d i c a t i n g u n r e s o l v e d h y p e r f i n e couplings with exchangeable protons. T h e g- a n d h y p e r f i n e - c o u p h n g tensors are typical of a C u ( I I ) ion having p s e u d o s q u a r e - p l a n a r or tetragonally-elongated octahedral coordination geometry. I n such a case the p e r p e n d i c u l a r p l a n e of these tensors c o r r e s p o n d s to the b a s a l c o o r d i n a tion p l a n e [22]. F r o m b o t h theoretical a n d m o d e l studies, the large value of the 14N s u p e r h y p e r f i n e splitting m the gll region c o r r e s p o n d s to the coup l i n g p e r p e n d i c u l a r to the C u - N b o n d . F o r such a s t r o n g l y c o u p l e d nitrogen the s u p e r h y p e r f i n e t e n s o r is e x p e c t e d to have n e a r l y axaal character, with the largest coupling, A~, as the unique value a s s o c i a t e d wtth a direction parallel to the C u - N b o n d , a n d a smaller coupling, A ~ , for directions n o r m a l to the b o n d [13,23] J4N and 1n E N D O R W h e n H 0 is set to the lowest-field ( m ic u = - 3 / 2 ) 65Cu h y p e r f i n e line (position A, Fig. 1) the E N D O R s p e c t r u m in Fig. 2a is observed. N o o t h e r r e s o n a n c e s are f o u n d at tlus field p o s m o n for

frequencies less t h a n 200 M H z . A s i n d i c a t e d in the figure, several r e s o n a n c e s are r o u g h l y s y m m e t r i c a b o u t n H (approx. 11.8 M H z ) a n d are assigned to p r o t o n s . T h e two higher frequency p e a k s at ap-

+4 N i

•

] F,

//~x.// /

/ N•

a /

\

I 10

I

I 14

I

I 18

Ml-lz

Fig 2 ]H and 14N ENDOR spectra near gll (field A, Fig 1) m 65CuTn (12CO3) with 100 kHz held modulation amphtude of approx 4 G (a) and approx 0 8 G (b) Peaks near 15 and 16 5 MHz are assigned to 14N, resonances symmetric about vH arise from protons Notice the changed phase of the mtrogen peaks (b), as well as the better resolution of proton signals Con&tions rmcrowave frequency, 9 565 GHz, nucrowave power, approx 20 #W, ra&ofrequency scan rate, 0 7 MHz/s, temperature, 2 K Arrow mdtcates free proton frequency

52 prox 14 6 and approx 16.3 are the Larmor-spht doublet from the strongly coupled mtrogen observed directly in the EPR spectrum. The absence of further sphttlngs Indicates that PI~ is small The nitrogen pattern is centered at AN~2= 15.4 MHz, which is in excellent agreement with the coupling constant measured by EPR, AN~2= 9.0/2 G, or 15.7 MHz [7]. A further confirmation of the 14N assignment is provided by the dependence of the E N D O R pattern on the 100 kHz field modulation amplitude. At modulation amplitudes below approx 1 G (Fig 2b), the proton resonances intensify relative to the nitrogen pattern and become better defined, and the nitrogen resonances appear as negative E N D O R peaks (decreases in the EPR Intensity). The detailed mechanisms responsible for this are not well understood [24] The proton signals observed in the gll region all have small hyperfme couplings A M< 3.5 MHz. It appears that none of these peaks is assignable to exchangeable protons (see below). Fig. 3 shows the proton and nitrogen E N D O R obtained when H 0 is set to the high-field portion of the EPR spectrum, in the vicinity of g ± . The two high-frequency features, which are associated

14N r--1 1H

~H

I

I

,

I

with strongly anlsotropic interactions (compare 3a, 3b) arise from exchangeable protons, as may be seen by comparmg spectra from dlcupnc transferrln in H 2 0 and in 2H20 , Fig. 4a and b, respectively. In spectra not shown, we find that these peaks shift appropriately with rmcrowave frequency so that each may be assigned to the resonance at v H + (Eqn. l) The two resulting coupling constants, A n, are 14.2 and 17.6 MHz, respectively The low-frequency partners, v H, for these resonances are expected to be less intense [17,18] and are not observed These proton couplings are about twice as great as the largest value observed in hexaquo-Cu(II) [25] However, in a protein-bound metal ion whose hgands include tyroslnate and hlstidme residues, extensive electron delocahzation might well account for the difference between 65CUE-transferrln-(IECO3)2 and the more ionic hexaquocopper complex. We cannot tell whether the resonances represent a single proton with large m-plane superhyperfine anlsotropy, or two inequivalent protons. For H 0 set in the vicinity of g-perpendicular, the E N D O R response between 12 and 18 MHz is a complex overlap of 14N and tH resonances, but examination of spectra taken under various conditions, over a range of magnetic fields, indicates that the peak at v = 16 8 MHz in Fig. 3 is associated with A N and corresponds to the frequency,

I

r~

J

/

/

\

I "\

~

\ '\\ 8 \

IH [

X• k

'

I

]

\

~'~-~ /

1 12

I

I 16

I

I 20

I

-

H=O

\

I 24

MHz

Fig 3 IH and 14N ENDOR spectra at (a) g± (field B) and (b) the highest field possible (field C) for A-site 65Cu2-transferrln(=2CO3)2 The resonancenear 16 8 MHz is the higher-frequency peak due to nitrogen, the lower-frequency peak (expected position approx 14 7 MHz) is obscured by the protons near vH at these field settings The indicated peaks above 18 5 MHz are assooated wtth exchangeable protons as described m the text Same condmons as Fig 2, except the radmfrequencyscan rate is now 4 MHz/s

=H20

I

I

I

I

16

18

20

22

MHz

Fig 4 ENDOR spectra of exchangeable proton resonances from 6SCu2-transfemn-(t2CO3)2 m (a) H20 and (b) 2H20 (field C, Fig 1) Same con&Uonsas Fig 2, except rmcrowave frequency ts 8 65 GHz

53

AN~2 + Vs. The lower frequency peak at A ~ / 2 v N is obscured by (Fig. 3a), or lost through crossrelaxation to, the proton resonances that are centered at v N --- 14.2 MHz (Fig. 3b) This assignment is confirmed by E N D O R spectra obtained at intermediate field values between gll and g± (not shown) and by the lack of a significant sluft in position when spectra are obtained at lower microwave frequency (Fig. 4). The simple InN E N D O R pattern at fields near g± indicates that there is little in-plane anlsotropy of the 14N hyperfine tensor; comparing the hyperfine couplings at gll and g± then shows that the superhyperfine tensor for the nitrogen hgand is nearly isotropic Similar behavior has been seen for the lrmdazole hgands of (Cu(lmldazole)4) 2+ [21], and the type I copper proteins, azurln, plastocyanin and stellacyanin [26] The value of the ~4N superhyperfine sphttmg in transferrln is considerably less than that in (Cu(inudazole)4) 2+ [21], and at the low end of the range reported for type I copper, although it is larger than that for Cu of cytochrome c oxadase [ 19,20]. We conjecture that the relatively low value reflects the influence of strong coordination of tyroslnate ion(s) to the copper [1]. Cu ENDOR Although the value of A~ u is easily obtainable from EPR, A cu is not, but it can be estimated with ENDOR. When the magnetic field is set to position C in the g-perpendicular region for a sample with isotopically enriched 65Cu, for frequencies above 25 MHz, the E N D O R response is a single broad peak centered at 44 MHz, w~th a width of

I

MHZ Fig 5 6SCu ENDOR spectrum 65Cu2-transfernn-(12CO3)2 (held C, FIg l) Virtually ldentacal signals are obtained tf 6SCu is incorporated only m the A-s~te Condluons, as m Fig 2

approx. 10 MHz (Fig. 5). The sample contammg 63Cu and 65Cu in natural abundance shows two broad, partially resolved E N D O R features, one at 44 MHz from 65Cu and one at 41 MHz, from 63Cu The center frequencies of the two features are in the ratio 44 M H z / 4 1 M H z = gN ( 6 3 C u ) / g N ( 6 5 C u ) , as expected for copper ENDOR. In general, as many as six E N D O R transitions for the copper nucleus may be observed (Eqn. 2). However, when H 0 is set either to the extreme low (gll) o r the the extreme lugh (g~_) field edges of the EPR envelope (fields A and C of Fig. 1), only a single copper hyperflne transition (m ! -- - 3 / 2 or rn~ = + 3/2, respectively) contributes to the END O R response. In each case only two E N D O R lines centered at ACU/2 (t = parallel or perpendicular) are expected from Eqn. 2 One interpretation of the sing.le peak in Fig. 5 would be that the two predicted resonances coalesce into a single observable feature. The measured E N D O R hnewidth then limits the maxamum pernusslble quadrupole interaction, p±Cu , to about 1 MHz. This value is anomalously small compared to Cu-O 4 model compounds, where pCu _ 5 MHz [27,28]. Thus, a second explanation wluch permits a more reasonable quadrupole couphng (Le., pCu__ 5 MHz), is preferred. If the 44 MHz peak is the htgher frequency peak of a two-line pattern involving such a quadrupole coupling, Eqn. 2 predicts a lower-frequency feature near 17 MHz and a corresponding value of about 30 MHz for AC~/2. Failure to observe the second lower-frequency resonance could result from a number of well-known mechanisms, including the lower intensity associated with the lower frequency [18] a n d / o r crossrelaxation with the mtrogens and protons absorbing near ttus frequency. Although we prefer the latter, either interpretation is consistent with the breadth of the gj. feature in the EPR spectrum. A direct determmatlon of Pll ( = - 2 P ± ) would confirm our tentative assignment, but the necessary frequency range is unavadable on our instrument.

Amon bmdmg Binding of Cu +2 to either of the two specific sites of transfernn in their native state reqmres the concomitant binding of an anion, ordinarily bicarbonate or carbonate. No E N D O R signals were

54

observed that could be assigned to 13C m the sample that contained taC-ennched CO g-, and thus the present ENDOR study offers no evidence for bicarbonate hgation directly to the copper mn of transferrin. The lack of a given set of ENDOR signals is of questionable stgnificance, due to the complexatles of the ENDOR response, and to the generally weaker intensity of low-frequency resonances It is mteresting to note that the pulsed EPR experiments also failed to detect nuclear modulation patterns from 13CO23 , although such signals were observed with 13C-enriched oxalate samples [8].

Stte equtvalences Although differences between the two metalbinding sites have been observed [1], the ENDOR spectra set tight limits on the extent of mequivalence in the copper-substituted protein A comparison of 65Cu ENDOR signals m A-site monocupric transfemn and dicuprlc transferrm suggests that the 65Cu hyperflne tensors for the two sites are equtvalent, or differ at most by a few tenths of a megahertz. Similarly, the relative sharpness of the 14N-ENDOR signals (Fig. 2a) from both singly and doubly substituted copper proteins m&cates that the properties of the nitrogenous hgand m the two sites are in&stingulshable at physiological pH. Apparent differences in the values of A N in the g-perpen&cular regmn reported m a study of single-site copper complexes of transferrln [10] were based on computer simulations of spectra, wtuch do not provide unique 'best fits', and therefore must g#ve way to the more direct measurements of the present report. Acknowledgements Ttus work was supported, in part, by Grants HL 12531 and AM 15056 from the National InsUtutes of Health, and PCM 7681304 from the National Science Foundatmn. Measurements were performed m the magnetic resonance facility of Northwestern University's Material Research Center, supported m part under the National Science Foundation NSF-MRL Program (Grant DMR 76-80847).

References l Chasteen, N D (1977)Coord Chem Rev 22, 1-36 2 Chasteen, N D , Wl~te, L K and Campbell, R F (1977) Biochemistry 16, 363-368 3 Price, E M and Gibson, J F (1972) J Blol Chem 247, 8031-9035 4 Aasa, R (1972)Btochem Blophys Res Commun 49, 806-812 5 Alsen, P, Aasa, R and Redfield, A G (1969) J Blol Chem 244, 4628-4633 6 Aasa, R and Alsen, P (1968)J Blol Chem 243, 2399-2404 7 Zweter, J L and Alsen, P ( 1 9 7 7 ) J Blol Chem 252, 6090-6096 8 Zweler, J L, Alsen, P, Pelsach, J and Mlms, W B (1979) J Blol Chem 254, 3512-3515 9 Fronclsz, W and Atsen, P (1982) Bloctum Blophys Acta 700, 55-58 10 Zweler, J L (1978)J Blol Chem 253, 7616-7621 11 Hoffman, B M , Roberts, J E, Kang, C H and Margohash, F (1981)J Blol Chem 256, 6556-6564 12 Roberts, J E, Hoffman, B M , Rutter, R and Hager, L P (1981) J Blol Chem 256, 2118-2121 13 Brown, T G and Hoffman, B M (1980) Mol Phys 39, 1073-1109 14 Past, G H and Hyde, J S (1968) J Chem Phys 49, 2449-2451 15 Post, G H and Hyde, J S (1970) J Chem Phys 52, 4633-4643 16 Roberts, J E, Brown, T G , Hoffman, B M and Pelsach, J (1980) J Am Chem Soc 102, 825-829 17 Abragam, A and Bleaney, B (1970) Electron Paramagnetlc Resonance of Transmon Ions, pp 87-94, 217-228, Clarendon Press, Oxford 18 Atherton, N M (1973) Electron Spin Resonance, pp 365-376, John Wiley, New York 19 Van Camp, H L, Wel, Y -H, Scholes, C P and King, T E (1978) Bloctum Blophys Acta 537, 238-246 20 Hoffman, B M , Roberts, J E, Swanson, M , Speck, S H and Margohash, E (1980)Proc Natl Acad So U S A 77, 1452-1456 21 Van Camp, H L, Sands, R H and Fee, J A (1981) J Chem Phys 75, 2098-2107 22 Goodman, B A and Raynor, J B (1970) Adv Inorg Chem Radlochem 13, 185-362 23 McGarvey, B R (1966) Transition Metal Chem 3, 89-199 24 Hayasba, Y and Mlyagawa, I (1977) J Magn Resonance 28, 3511-364 25 Atherton, N M and Horsewdl, A J (1979) Mol Phys 37, 1349-1360 26 Roberts, J E (1981) Ph D Thesis, Northwestern Umverslty, Evanston, IL 27 Wlute, L K and Belford, R L (1976) J Am Chem Soc 98, 4428-4438 28 Wlute, L K and Belford, R L (1976)Chem Phys Lett 37, 553-557