1H-NMR studies of structural homologies between the heme environments in horse cytochrome c and in cytochrome c-552 from Euglena gracilis

307

Biochimica et Biophysica Acta, 668 (1981) 307--320

© Elsevier/North-Holland Biomedical Press

BBA 38666 1H-NMR STUDIES OF STRUCTURAL HOMOLOGIES BETWEEN THE HEME ENVIRONMENTS IN HORSE CYTOCHROME c AND IN CYTOCHROME c-552 FROM E U G L E N A G R A C I L I S

REGULA M. KELLER and KURT WUTHRICH Institut fiir Molekularbiologie and Biophysik, Eidgen6ssische Technische Hochschule, 8093 Ziirich-H6nggerberg (Switzerland)

(Received November 19th, 1980) Key words: Cytochrome c conformation; Heme crevice; Structure homology. 1H-NMR; (Euglena gracilis)

Summary With the use of proton-proton Overhauser enhancement experiments the spatial arrangement relative to the heme group of amino acid side chains in the heme crevice of horse ferrocytochrome c and ferrocytochrome c-552 from Euglena gracilis was investigated. From these data and the known crystal structure for mammalian cytochromes c, individual assignments were obtained for several aromatic residues in horse ferrocytochrome c. This then provided a basis for delineating homologies between the polypeptide conformations near the heme group in horse ferrocytochrome c and ferrocytochrome c-552, for which no crystal structure has as y e t been described.

Introduction It is by now quite generally acknowledged that correct alignment of the amino acid sequences of homologous proteins can be greatly aided by data on the spatial folding of the polypeptide chains [1]. This was nicely illustrated by the alignments obtained for cytochromes of the c-type which differ by extensive deletions and insertions in the polypeptide chains [2--4]. As a complementation of the information available from the amino acid sequences and, where applicable, from the X-ray structures, spectroscopic data may further add to the characterization of structural homologies [ 5--10]. We have recently mainly concentrated on comparisons of the coordination geometry at the heme iron and the electronic structure of ferric heme c in different homologous cytochromes c [11--14]. In the present papers these studies were extended to

308 amino acid side chains near the heme. Homologies between the spatial s t r u c tures of the heme crevices in horse cytochrome c and cytochrome c-552 from Euglena gracilis, for which no crystal structure is as yet available, were delineated. The conclusions on structural homologies between horse c y t o c h r o m e c and cytochrome c-552 are based on studies of selective nuclear Overhauser effects [15--17] between protons of heme c and nearby amino acid side chains. For both proteins identification of the spin systems of several amino acids in the heme crevice, and in some cases assignment to a particular position in the amino acid sequence, is described. Materials and Methods Cytochrome c-552 from E. gracilis was isolated and purified as described previously [18]. For the NMR studies, cytochrome c-552 solutions in 0.05 M deuterated phosphate buffer, p2H 7.0, were prepared. Horse heart ferricytochrome c 'Type VI' was obtained from Sigma and dissolved in 2H20 which also contained 0.1 M NaC1. The p2H was adjusted by addition of minute amounts of NaO2H and 2HC1. Solutions of the reduced proteins were obtained by addition of small amounts of solid Na2S204. High resolution 1H-NMR spectra were recorded in the Fourier mode on a Bruker HX-360 spectrometer. Chemical shifts are expressed in parts per million (ppm) from internal sodium 3-trimethylsilyl[2,2,3,3-2H]propionate. Truncated-driven nuclear Overhauser difference spectra were recorded as described previously [15]. To further clarify some connectivities between aromatic ring protons, a two-dimensional nuclear Overhauser enhancement spectrum of horse ferrocytochrome c was recorded. This experiment used the following, previously described [19,20] sequence of three non-selective 90 ° pulses: (90°-tl-90 °-rm-90°-t2). tl is the evolution period, rm the mixing time and t2 the observation period [20]. 512 experiments with different, equidistant values of tl were performed, whereby n transients were accumulated for each value of t~ in order to obtain a workable signal-to-noise ratio. Additional details are given in the legend to Fig. 7. Results and Discussion

New resonance assignments in ferrocytochrome c-552 Besides the axial histidine of the heme iron, c y t o c h r o m e c-552 from E. gracilis contains eight aromatic amino acid residues, i.e. two tryptophans, one phenylalanine and five tyrosines [18]. In ferrocytochrome c-552 the spin systems of the two tryptophans and of four tyrosines were previously identified at 29°C [21]. The identification of the remaining two aromatic spin systems is described in the following, together with new data on the locations of selected aromatic rings relative to the heme group (Fig. 1 ). Trace A of Fig. 2 shows the aromatic region of the ferrocytochrome c-552 spectrum at 77°C. The spin systems of the fifth tyrosine, which was arbitrarily

309

Z

o

Fe

2

/

Fig. 1. D r a w i n g s o f t h e h e i n e g r o u p a n d t h e axial ligands for c - t y p e c y t o c h r o m e s b a s e d o n t h e a t o m i c c o o r d i n a t e s o f t u n a f e r r o c y t o c h r o m e c o b t a i n e d f r o m t h e p r o t e i n d a t a b a n k . L e f t : View p e r p e n d i c u l a r t o t h e h e i n e p l a n e f r o m t h e side o f t h e axial m e t h i o n i n e . T h e ~-pyrrole p o s i t i o n s are n u m b e r e d f r o m 1 to 8 a n d t h e m e s o p o s i t i o n s f r o m ~ to 6. Right: V i e w parallel t o t h e h e m e p l a n e in t h e d i r e c t i o n f r o m t h e r n e s o p o s i t i o n 6 t o t h e m e s o p o s i t i o n ~.

, ' ', ~

",1

t

,!i

tt

P'L

Lt

•

• ~

-

,

,I J'Ii,,,t

'

~I

I i

i, I

,

,'

/

~''°""ITy~RV .......... ,.

PHE-6

J

I

I

I

7.5

7

6.5

6

PPM

Fig. 2. ( A ) S p e c t r a l r e g i o n f r o m 5.9 t o 7.6 p p m o f t h e 3 6 0 M H z 1 H - N M R s p e c t r u m of a 0 . 0 0 3 M s o l u t i o n o f f e r t o c y t o c h r o m e c - 5 5 2 , p 2 H 7.5, T 7 7 ° C . T h e r e s o l u t i o n w a s i m p r o v e d b y m u l t i p l i c a t i o n of t h e free i n d u c t i o n d e c a y w i t h a p h a s e - s h i f t e d sine bell [ 3 0 ] . T h e spin s y s t e m s of t h e single p h e n y l a l a n i n e in posit i o n 6 a n d of T y r V are i n d i c a t e d . ( T h e n u m e r a t i o n of t h e T y r spin s y s t e m s I - - V [ 2 1 ] is a r b i t x a r y . T h e y h a v e n o t b e e n i n d i v i d u a l l y assigned.) (B) I d e n t i f i c a t i o n of t h e Phe 6 spin s y s t e m . I r r a d i a t i o n a t 6.75 p p m ( o p e n a r r o w ) r e s u l t e d in t h e collapse of a t w o - p r o t o n d o u b l e t a t 7.29 p p m a n d a o n e - p r o t o n t r i p l e t a t 6 . 5 3 p p m (filled a r r o w s ) . Since t h e r e s o n a n c e of Phe 6 a t 6.75 p p m o v e r l a p s w i t h a t w o - p r o t o n d o u b l e t o f t h e p r e v i o u s l y i d e n t i f i e d spin s y s t e m of T y r I I I [ 2 1 ] , t h e s e c o n d d o u b l e t of T y r I I I a t 7.08 p p m w a s simultaneously decoupled.

310

itl'~ ~ ~

B

~

I

~t~

1

I

f

I

I

7.5

7

6.5

6

PPM

Fig. 3. 1 H - N M R s t u d i e s o f t h e s p i n s y s t e m o f t h e p a r t i a l l y i m m o b i l i z e d r i n g o f Phe 6 in t h e f e r r o c y t o c h r o m e c - 5 5 2 s o l u t i o n o f Fig. 2; T 2 6 ° C . ( A ) S p e c t r a l r e g i o n f r o m 5.9 t o 7.6 p p m . (B a n d C) D o u b l e resonance irradiation of the broad multiplets at 6.41 and 7.08 ppm (open arrows) leads to decoupling of t h e t w o - p r o t o n d o u b l e t a t 7 . 3 2 p p m (filled a r r o w s ) . T h e s p e c t r a l r e s o l u t i o n w a s i m p r o v e d b y m u l t i p l i c a t i o n o f t h e free i n d u c t i o n d e c a y w i t h a p h a s e - s h i f t e d sine bell [ 3 0 ] .

named Tyr V, and the single phenylalanine are indicated. Tyr V gives rise to two sharp doublets for which the connectivity corresponding to an AA'XX' spin system could be established by spin decoupling. Trace B of Fig. 2 shows that irradiation at 6.75 p p m causes the simultaneous collapse of a two-proton d o u b l e t at 7.29 ppm and a one-proton triplet at 6.53 ppm. The interpretation is that the resonance of the C3,5-protons of Phe 6 is at 6.75 ppm and that the doublet at 7.29 ppm and the triplet at 6.53 ppm correspond to the C2,6protons and the C4-proton, respectively. The resonance at 6.75 ppm of the C3,5-proton overlaps with a previously assigned Tyr doublet and is not resolved. Additional observations (Fig. 3) further support this interpretation, i.e. at 26~C two separate lines in symmetrical positions relative to 6.75 ppm were found to correspond to the C3,5-protons of Phe 6. The exchange of the t w o protons between the respective spatial locations in the protein is, however, still sufficiently rapid at 26°C SO that the C2,6-proton doublet is fully decoupled by irradiation at either of the t w o positions. For Phe 6 a transition from an asymmetric AA'MRX type spectrum to a symmetric AA'MM'X spectrum [22] was thus observed, with limited mobility a b o u t the C~--C ~ bond throughout the temperature range from 26 to 77°C. Approximate ring flip frequencies (Table I) were obtained at 26°C from saturation transfer between the two C3,5-proton lines and at 77°C from the line shape. Restricted rotational mobility is clearly manifested also for Tyr V, wher~ the sharp doublets seen at 77°C (Fig. 2) correspond to broad, featureless resonances at 26°C (Fig. 3). Fig. 4 provides quite detailed data on the relative locations in space of heme c and the previously identified ABCD spin system of 'Trp II' [21], which corresponds either to Trp 59 or to Trp 83. In truncated-driven Overhauser enhancement difference spectra recorded with pre-irradiation of the meso-proton ~ at 9.96 ppm, the strongest peaks correspond to the resonances A and B of the six-

311

T(sec) 2.0

A

B

X

~

,,

,, IRRAD(PPM)

~

, ~ v v ~ / ~ L

9.96

i

I I

4.0

1.0

t

I1il

~/"~"~'~ " < ~ "

~'~ i! ~'~V

Ii

Ii

I '

~

~

>'2.76

• 0.25

I 8.5

t

I 7.5

I

I 6.5 PPM

Fig. 4. T r u n c a t e d - d r i v e n n u c l e a r O v e r h a u s e r e n h a n c e m e n t d i f f e r e n c e s p e c t r a r e c o r d e d in t h e f e r r o c y t o c h r o m e c - 5 5 2 s o l u t i o n o f Fig. 2; T 3 0 ° C . T h e p r e - i r r a d i a t i o n t i m e s are listed o n t h e left. T h e pre-irradiat i o n f r e q u e n c i e s i n d i c a t e d o n t h e r i g h t of e a c h t r a c e c o r r e s p o n d t o the h e m e m e s o - p r o t o n ~ a t 9 . 9 6 p p m a n d t h e M e t 56 m e t h y l g r o u p a t - - 2 . 7 6 p p m . T h e p e a k s i n d i c a t e d w i t h A a n d B w e r e p r e v i o u s l y assigned t o t h e spin s y s t e m T r p n [ 2 1 ] , t h e p e a k X c o r r e s p o n d s t o t h e slnglet r e s o n a n c e o f o n e of t h e t w o t r y p t o p h a n s in c y t o c h r o m e c - 5 5 2 [ 2 1 ] .

membered ring of Trp II. Upon pre-irradiation of the methyl protons of the axially coordinated Met 56 at --2.76 ppm, the strongest effect is seen on one of the t r y p t o p h a n singlet resonances [21], and weaker effects occur for the resonances A and B of Trp II. These experiments thus imply that the singlet resonance at 6.95 ppm comes from Trp II. They further provide, together with additional nuclear Overhauser data, the following information on the spatial location of the indole ring of Trp II relative to heme c: the periphery of the six-membered ring is near meso-proton e and ring methyl 3, the five-membered ring is near the methyl group of Met 56, and the thioether methyl 2 of heme c is above the plane of the indole ring. Model building with CPK space-filling models (Corey-Pauling atomic models with new connectors by Koltun), which took into account this distance information as well as the previously established methionine coordination geometry [14], suggested that the indole ring plane is nearly perpendicular to the heme plane and that the indole nitrogen is in close proximity to the iron-bound sulphur atom of Met 56. This location of the indole ring would also be compatible with the chemical shifts of the heme resonances, i.e. downfield shifts relative to the isolated heme of approx. 0.5 ppm for meso-proton a and ring methyl resonance 3, and an upfield shift of approx. 1.5 ppm for the thioether methyl 2. Cytochrome c-552 contains a total of 50 m e t h y l groups from ten alanines, 10 valines, three leucines, four isoleucines, five threonines and one methionine [18]. In the reduced protein only three of the 50 methyl resonances are at higher field than 0 ppm [9]. One of these corresponds to the axially coordinated methionine [9]. The other two high-field resonances are doublets which

312

2,6

3,5 4 T(sec) 20

~

I

I

I

I

i

I it

/'~/,

~ I '~/'~,

IRRAD (PPM)

]11 I ~

9.32

Iq' II Ill

10

i :

0.25

I

I

I

I

II t Jl ]

-328

I

0.05 I 75

I 7.0

I 6.5

PPM

Fig. 5. T r u n c a t e d - d r i v e n n u c l e a r O v e r h a u s e r e n h a n c e m e n t d i f f e r e n c e s p e c t r a i n a 0 . 0 0 8 M s o l u t i o n o f ferr o u s h o r s e h e a r t c y t o c h r o m e c, p 2 H 6 . 4 , T 3 0 ° C . T h e r e s o n a n c e s o f a p r e v i o u s l y i d e n t i f i e d [ 2 3 ] p h e n y l alanine spin system are indicated by the numbers of the ring'atoms, The preirradiation times are listed on t h e left. T h e p r e - i r r a d i a t i o n f r e q u e n c i e s i n d i c a t e d o n t h e r i g h t c o r r e s p o n d t o t h o s e o f t h e h e m e mesoproton ~ at 9.32 ppm and the Met 80 methyl protons at --3.28 ppm.

could be simultaneously decoupled by irradiation at 0.08 ppm. Since no difference decoupling pattern typical for the valine Ca proton could be detected, these resonances were assigned to the two 5 methyls of one of the three leucines in cytochrome c-552. N e w resonance a s s i g n m e n t s in horse f e r r o c y t o c h r o m e c

Numerous individual assignments of aromatic and aliphatic resonances in horse ferrocytochrome c were recently described [23,24]. These assignments were based primarily on comparisons of homologous cytochromes c and on ring current calculations using the crystallographic atom coordinates. The following nuclear Overhauser enhancement data were used to obtain independent checks of the previously proposed resonance assignments. Fig. 5 shows truncated-driven nuclear Overhauser enhancement difference spectra of horse heart ferrocytochrome c obtained, respectively, with preirradiation of the resonances of the Met 80 methyl protons and the heme mesoproton ~. The peaks identified in the figure correspond to a phenylalanine spin system which was previously assigned to Phe 10 [23]. Upon pre-irradiation of the Met 80 methyl line the peaks emerged in the order C2,6H, C3,5H and C4H. Upon preirradiation of the heme m e s o - p r o t o n ~, the three peaks appeared simultaneously. On the basis of the X-ray structures for mammalian cytochromes c [25] these observations imply that this spin system corresponds to Phe 82.

313

TrpI

8

i

11

!l

Phe-6

ii

Phe_lO

5

t

I~ I

10

I

I

I

i

?

.L I I

I)I

5

,

I

t ~l',l' ~ t, I

I

I

I

0

I

I

I

[

PPM

F i g . 6. ( A ) T r u n c a t e d - d r i v e n n u c l e a r O v e r h a u s e r e n h a n c e m e n t d i f f e r e n c e s p e c t r u m o f f e r r o u s h o r s e h e a r t c y t o c h r o m e c o b t a i n e d w i t h p r e - i r r a d i a t i o n d u r i n g 2 s o f t h e L e u 32 m e t h y l r e s o n a n c e a t - - 0 . 7 5 p p m , T = 5 0 ° C . T h e s a m e p r o t e i n s o l u t i o n w a s u s e d as in F i g . 4. (B) T r u n c a t e d - d r i v e n n u c l e a r O v e r h a u s e r e n h a n c e m e n t d i f f e r e n c e s p e c t r u m o f f e r r o c y t o c h r o m e c - 5 5 2 f r o m E. gracilis o b t a i n e d w i t h p r e - i r r a d i a t i o n o n t h e h i g h - f i e l d m e t h y l d o u b l e t at - - 0 . 6 1 p p m [ 9 ] , T 3 0 ° C . T h e s a m e p r o t e i n s o l u t i o n w a s u s e d as in F i g . 2. I n b o t h t r a c e s s p i n s y s t e m s o f a r o m a t i c r i n g s a r e i d e n t i f i e d . T h e a d d i t i o n a l n u m b e r s a n d t h e g z e e k lett e r s i n d i c a t e r e s o n a n c e s o f h e i n e c a c c o r d i n g t o t h e n u m e r a t i o n in F i g . 1.

Trace A of Fig. 6 shows a truncated-driven nuclear Overhauser enhancement difference spectrum obtained with preirradiation of one of the high-field shifted methyl doublets which were previously assigned to Leu 32 [24,26]. Based on the X-ray structure of mammalian c y t o c h r o m e c [25] the nuclear Overhauser effects for the ring methyls 1 and 8 and the meso-proton 5 provide further support for the assignment of the Leu 32 methyl lines. From similar arguments the phenylalanine spin system in Fig. 6A must correspond to Phe 10. This contrasts with a previous assignment to Phe 82, which was based on comparison of homologous cytochromes c [10] * Horse heart cytochrome c contains four tyrosine residues in positions 48, 67, 74 and 97. So far two tyrosine spin systems were identified in the reduced protein and assigned to Tyr 48 and Tyr 74 [23]. The spin system which was assigned to Tyr 48 is of particular interest, since it corresponds to a tyrosine ring with restricted rotational mobility [27,28]. We recorded a two-dimensional nuclear Overhauser enhancement experiment [19,20] (Fig. 7) to further check on this assignment. In the contour plot of the aromatic region shown in Fig. 7 the peaks on the diagonal correspond to the normal one-dimensional 1H-NMR spectrum. Selective transfer of magnetization through nuclear Overhauser enhancement or chemical exchange is manifested b y cross-peaks which connect individual diagonal peaks. Each cross-peak appears twice in symmetrical positions with respect to the diagonal peaks [20]. Obviously, the spectral * I n a private c o m m u n i c a t i o n D r . G . R . M o o r e i n f o r m e d us t h a t he a n d h i s c o l l e a g u e s h a v e r e c e n t l y also f o u n d e v i d e n c e ~ o m n u c l e a r O v e r h a u s e r e x p e ~ - n e n t s t h a t t h e p r e v i o u s a s s i g n m e n t s of P h e 10 a n d P h e 82 m u s t b e i n t e r c h a n g e d [ 3 2 ] .

314 1

I

I ,

6-

c

~

"

i

-

I

I

I

8

7

6

-

60 z (PPM) Fig. 7. C o n t o u r plot o f the spectral region f r o m 5.1 to 8.4 ppm o f a two-dLmen~on ~1 nuclear Overhauser e n h a n c e m e n t 1 H - N M R s p e c t r u m at 3 6 0 MHz of h o r s e h e a r t f e r r o c y t o c h r o m e c. T h e s p e c t r u m w a s r e c o r d e d in a 0 . 0 1 2 M s o l u t i o n o f t h e p r o t e i n in 2 H 2 0 , p 2 H 7.0, T 2 5 ° C . T h e s p e c t r a l w i d t h w a s 5 4 9 5 Hz, t h e d a t a file c o n s i s t e d o f 5 1 2 p o i n t s in b o t h the e I a n d ¢o2 d i r e c t i o n s , t h e m i x i n g t i m e w a s 1 0 0 ms, f o r e a c h v a l u e o f t 1 6 4 t r a n s i e n t s w e r e a c c u m u l a t e d , a n d t h e t o t a l a c c u m u l a t i o n t i m e was a p p r o x . 20 h. B e f o r e F o u r i e r t r a n s f o r m a t i o n t h e free i n d u c t i o n d e c a y s w e r e m u l t i p U e d in b o t h d i m e n s i o n s w i t h a ~ / 6 4 p h a s e - s h i f t e d sine bell [ 3 0 ] . T h e s p e c t r u m was p l o t t e d in t h e a b s o l u t e v a l u e p r e s e n t a t i o n . T h e crosses i d e n t i f y t h e c r o s s - p e a k s w h i c h m a n i f e s t t h e c o n n e e t i v i t i e s b e t w e e n t h e p r o t o n s of a s l o w l y flipping t y r o sine ring (see t e x t ) . T h e e n t i r e s p e c t r u m f r o m - - 4 . 0 to 10.0 p p m w a s p r e s e n t e d e l s e w h e r e t o g e t h e r w i t h a d d i t i o n a l e x p e r i m e n t a l details [ 31 ].

region in Fig. 7 manifests exclusively exchange of magnetization between aromatic protons with chemical shifts from 5.1 to 8.4 ppm. Furthermore, since the experiment used a short mixing time, sizeable nuclear Overhauser effects were expected only for very closely spaced protons, such as neighbouring protons on the same aromatic ring [20]. In Fig. 7 six pairs of cross-peaks which connect the four diagonal peaks of the ABCD spin system of a slowly rotating tyrosine ring are identified by crosses. Inspection of the peaks located on straight lines at 5.55 ppm parallel to the a~l and ~2 axes shows that the four components of this spin system are at 5.55, 6.60, 6.73 and 7.17 ppm. The combined effects of exchange through the ring flips about the C~--C ~ bond [22] and nuclear Overhauser enhancement between C2H and C3H, and C5H and C6H, respectively, lead to exchange of magnetization among all four ring protons. The assignment of the peak at 6.60 ppm, which is in the one-dimensional spectrum overlapped with a two-proton doublet of a different tyrosine ring [23], was further confirmed by spin decoupling. The three resonances at 5.55, 6.73 and 7.17 ppm of the tyrosine spin system identified in Fig. 7 correspond to lines which were previously assigned to Tyr

315 48 [23]. The previous erroneous identification of the fourth resonance at 6.3 p p m did n o t affect the parameters which characterize the ring mobility b u t affected the simulation of the temperature
BC

T(se, 2.0

1.0

~/~

I,I,

I ~

llll

"1 /I I, ,~

/I

o.2

0.1 ~-~?--~J, I 75

~ i

I 6.5

i

i 5.5

PPM

Fig. 8. T r u n c a t e d - d r i v e n nuclear Overhauser enhancement difference spectra recorded in the horse ferrocytoehrome c s o l u t i o n o f Fig. 7 a t 2 0 ° C w i t h p r e - i r r a d i a t i o n o f t h e t y r o s i n e r e s o n a n c e a t 5 . 5 5 p p m f o r d i f f e r e n t t i m e s as i n d i c a t e d o n t h e l e f t . T h e f o u r r e s o n a n c e s o f t h e t y r o s i n e A B C D spin s y s t e m i d e n t i f i e d in Fig. 7 are l a b e l l e d . T h e a r r o w p o i n t s t o t h e p e a k w h i c h h a d p r e v i o u s l y b e e n a s s i g n e d t o r e s o n a n c e C

[283.

316

chrome c, i.e. Tyr 48, Tyr 67 and Tyr 47, are far away from Phe 10 on the opposite side of the heme plane. Assignment to Tyr 97 was recently also suggested on the basis of ring current calculations [29]. Homology between the heme crevices o f horse ferrocytochrome c and ferrocytochrome c-552 The individual assignments of the 1H-NMR lines for heme c and the axial ligands of the heme iron in both horse ferrocytochrome c [11,13] and ferrocytochrome c-552 [14], which provide the reference points in space for studies of the location of amino acid side chains in the heme crevice, were obtained entirely from NMR experiments and without use of data on the protein conformation obtained by other methods. For horse ferrocytochrome c they therefore provided the basis for a valid comparison of the conformations in single crystals and in solution. The locations of selected amino acids in the crystal structure are shown in Fig. 9. The proton-proton Overhauser effects expected between the residues in Fig. 9 have nearly all been observed, as was described in more detail above. Spatial proximity between the heme c meso-proton a, e-CH3 of Met 80 and the aromatic ring of Phe 82 was manifested in the experiments of Fig. 5. The data in Fig. 6A clearly indicated that the methyl groups of Leu 32 are at similar distances from the ring of Phe 10 and the heme edge with the H-carbons 1 and 8 and the meso-carbon 5. The data of Figs. 7 and 8 manifested the close proximity of Phe 10 and Tyr 97. In the three-dimensional structure the distances between Trp 59 and heme c or the axial Met are approx. 7.0 A and thus longer than they appear from the projection in Fig. 9. No Overhauser

~59 F82

H18~

.0=~ 3

L32 F10

F i g . 9. C o m p u t e r d x a w i n g o f t u n a f e r r o e y t o c h r o m e c b a s e d o n the a t o m i c c o o r d i n a t e s f r o m the p r o t e i n data b a n k . S h o w n are h e i n e c, the axial ligands o f the h e i n e iron and five a d d i t i o n a l a m i n o side c h a i n s w h i c h w e r e u s e d for the s t u d i e s o f h o m o l o g i e s b e t w e e n m a m m a l i a n c y t o c b x o m e s c and c y t o c b x o m e c - 5 5 2 . T h e c o m p l e t e s t r u c t u r e o f h e i n e c is s h o w n , w i t h o u t h y d r o g e n a t o m s and w i t h -C-C,- f r a g m e n t s in the p o s i t i o n s 2 and 4 . T h e c o m p l e t e a m i n o acid side c h a i n s a r e s h o w n , i n c l u d i n g t h e C a s b u t w i t h o u t h y d r o g e n a t o m s . T h e structure is v i e w e d n e a r l y parallel to t h e h e m e plane and a l o n g a line t h r o u g h a p o i n t h a l f - w a y b e t w e e n t h e m e s o - c a r b o n ~ and t h e H-carbon 3, and t h r o u g h the h e i n e i r o n ( F i g . 1).

317 effects were observed between Trp 59 and heme c or Met 80. For His 18 Overhauser effects with preirradiation of Leu 32 were observed in experiments at 20 and 37°C, which were recorded with higher irradiation power than Fig. 6A. In ferrocytochrome c-552, where no X-ray structure has as y e t been obtained, Overhauser effects with specified protons of heme c and/or the axial ligands indicated that some of the identified spin systems come from residues in spatial locations which correspond closely to those of the amino acids of horse ferrocytochrome c shown in Fig. 9. Thus the data for Trp II in Fig. 4 coincide with those for Phe 82 of horse ferrocytochrome c in Fig. 5. Similar to the horse protein the spectrum of ferrocytochrome c-552 contains two highfield shifted methyl resonances of leucine between 0 and --1 ppm [9]. The Overhauser effects obtained with pre-irradiation on one of these lines (Fig. 6B) include peaks corresponding to the heme c methyls 1 and 8, heme c m e s o proton 6, His 14, Phe 6 and Trp I. These data place the Leu methyl groups near the heme edge with ~-carbons 1 and 8 and m e s o - c a r b o n 5, the axial His and Phe 6, which would be compatible with locations of these three residues in similar positions as Leu 32, His 18 and Phe 10 in horse ferrocytochrome c (Fig. 9). In an additional experiment the C2,6-protons of Phe 6 were pre-irradiated. Overhauser effects were observed for the heme m e s o - p r o t o n 5, the heme ring m e t h y l 1, the axial His, the high-field shifted Leu methyl lines, Tyr V and Trp I. This would again be compatible with the assumption that the spatial arrangements of His 14, a leucine, Phe 6 and Tyr V relative to heme c in ferrocytochrome c-552 are similar to those of His 18, Leu 32, Phe 10 and Tyr 97 in Fig. 9. The structural similarities manifested in the NMR data include further the internal mobility of the aromatic rings. For Phe 10 and Tyr 97 in horse ferrocytochrome c as well as for Phe 6 and Tyr V in cytochrome c-552 restricted rotational mobility was clearly evidenced (Figs. 2, 3, 7 and 8) [28]. Table I lists the NMR parameters of the amino acid residues or spin systems for which the proton-proton Overhauser effects indicated similar spatial locations relative to the heme. Overall the homologies of conformation indicated by Table I coincide well with the homologies suggested by previously published sequence alignments [2--4]. For the spin systems in ferrocytochrome c-552 which were not individually assigned by NMR experiments, the assignments which correspond to the sequence homologies are indicated in Table I. For one amino acid residue, where two different alignments were suggested [3,4], it appears that the discrepancy can be resolved with the NMR data. The discrepancy between different sequence alignments concerns Leu 32 in horse cytochrome c and Leu 27 in cytochrome c-552. Dickerson and coworkers [2,3] suggested that the locations of these two residues differed by three residues in the sequence alignments, whereas Schwartz and D a y h o f f [4] suggested homologous locations. The NMR data clearly support that the two leucines are homologous in the spatial structure. For the other residues in Table I, with the exception of the last mentioned Trp 59 and Trp I (see below), the indications of homology from the different sources coincide. For Met 80 and Met 56 it was previously shown that they are bound to the heme iron with the same chirality [14]. With regard to the homology between Phe 82 and Trp 59, it is worth noting that Trp 59 is replaced by phenylalanine in all other cytochromes f [3,4]. For the aromatics Phe 10 and Tyr 97 in horse cytochrome c and Phe 6

318 TABLE I CHEMICAL SHIFTS, 5, IN ppm OF AMINO ACID SIDE CHAINS IN HORSE HEART FERROCYTOC H R O M E c A N D F E R R O C Y T O C H R O M E e - 5 5 2 F R O M E. G R A C I L I S H o m o l o g y in t h e s p a t i a l s t r u c t u r e s o f t h e t w o p r o t e i n s is d i s c u s s e d in t h e t e x t . F o r t h e a r o m a t i c r i n g s o f p h e n y l a l a n i n e a n d t y r o s i n e t h e r o t a t i o n a l m o b i l i t y a b o u t t h e C ~ C 7 b o n d [ 2 2 ] is also i n d i c a t e d . ppm Horse fe~ocytochrome

c

Met 80 --3.28 --3.73 --1.87 --2.58 ---0.19 3.21

5(eCH3) 6 (7'CH) 6 (~,CH) 6 (~'CH) 6 (~CH) 6 (~CH) 6(C2, 6H) 6(C3, 5H) 6 (C2H) 6 (C4H) 6 (C5H) 5 (C6H) 6(C7H) Ring mobility

Phe 82 6.71 7.40 7.25

Leu 32 b 0.53 --0.59 --0.75

6 (~CH) 6(61CH3) 6(62CH3) 6 (C2, 6H) 6(C3,5H) (

Met 56 --2.76 --3.1 --1.2 --3.1 --0.3 3.0 T r p II ( T r p 597 a

6.95 8.36 7.89 7.39 7.47

Only symmetric spectrum of mobile ring seen His 18 b 0.50 0.13

6(C2H) 6(C4H)

Ferrocytochrome c-552

Phe 10 7.12 6.2 7 . 1 } 6,7 c

His 14 1.05 0.27 Leu (Leu 27) a 0.08 --O.61 --0.86 Phe 6 7.32 6.41 7.08}6.75

6 (C4H)

6.28

6.57

Ring mobility d

k(20°C) ~ 60 /z(50°C) ~ 3 0 0 0

k(26°C) ~ 10 k(77°C) ~ 1000

Tyr 97 7.17 6.60 ~ 6"78c

Tyr V (Tyr 74) }6.94

6.73 5.55 } 6.18 c k(20°C) = 4 k(50°C) = 100

}6.32 B e l o w 5 0 ° C line b r o a d e n i n g indicates restricted mobility

6(A) 6(B) 6(C) 6(D) Ring mobility d

6 (C2H) 8(C4H or 6(C5H or 6 (C6H or 6 (C7H or

C7H) C6H) C5H) C4H)

T r p 5 9 b,e 6.99 7.10 5.74 6.71 7.60

Trp I (Trp 83) e 6.59 6.87 5.48 6.35 7.54

a I n d i v i d u a l a s s i g n m e n t s i n d i c a t e d in p a r e n t h e s e s are b a s e d o n h o m o l o g y a r g u m e n t s (see t e x t ) . b Assignments and NMR parameters for these residues are from Refs. 23 and 24. c S m a l l d i f f e r e n c e s b e t w e e n t h e s e c h e m i c a l s h i f t s a n d t h e c o r r e s p o n d i n g d a t a in T a b l e I o f R e f . 2 3 are probably due to p2H differences between the samples used. d 1% t h e f r e q u e n c y o f r i n g flips a b o u t t h e C ~ - - C 7 b o n d a t t h e t e m p e r a t u r e i n d i c a t e d . e T h e s e r e s i d u e s are i n c l u d e d h e r e side b y side e v e n t h o u g h t h e r e is n o e v i d e n c e t h a t t h e y s h o u l d b e in h o m o l o g o u s l o c a t i o n s in t h e s e q u e n c e s or t h e c o n f o r m a t i o n s o f t h e t w o p r o t e i n s (see t e x t ) .

319

and Tyr 74 in cytochrome c-552 it seems worthwhile to re-emphasize that the conformational homology includes the restricted rotational mobility of the rings. Trp 59 in horse cytochrome c and Trp I in cytochrome c-552 (which must be Trp 83 since there are only two Trp per molecule) are listed in Table I, even though there is no evidence for homology from either sequence alignment or NMR. From the X-ray data on mammalian cytochromes c [2] and the NMR data of Fig. 6 these tryptophans must be on different sides of the heme plane in the two proteins, i.e. in contrast to Trp 59 in Fig. 9 Trp 83 in cytochrome c-552 must be on the same side as the axial His. The NMR observations thus lead to the conclusion that the C-terminal segment of the polypeptide chain of cytochrome c-552 is folded back towards the heme. It may be added that according to the above-mentioned sequence alignments, position 80 in cytochrome c-552 corresponds to the C-terminus in mammalian cytochrome c [2--4]. Finally, it is quite instructive to realize that while the similarity of the chemical shifts for Trp 59 in horse cytochrome c and Trp 83 in cytochrome c-552 (Table I) indicates similar locations of the indole rings in the highly symmetrical ring current field of the heme [22], the locations of the rings in the two molecules must be largely different.

Acknowledgements We would like to thank Professor A. Schejter for a gift of cytochrome c-552, Dr. Anil Kumar for recording the two-dimensional NOE spectrum and the Swiss National Science Foundation for financial support (project No. 3.528,79). References 1 2 3 4 5 6

7 8 9 10 11 12 13 14 15 16 17 18 19 20

Schulz, G.E. a n d S c h i r m e r , R.H. ( 1 9 7 9 ) Principles o f P r o t e i n S t r u c t u r e , Springer-Verlag, New Y o r k D i c k e r s o n , R.E., T i m k o v i c h , R. a n d Almassy, R.J. ( 1 9 7 6 ) J. Mol. Biol. 1 0 0 , 4 7 3 - - 4 9 1 D i c k e r s o n , R.E. ( 1 9 8 0 ) Sci. A m . 2 4 2 , 9 8 - - 1 1 0 S c h w a r t z , R.M. a n d D a y h o f f , M.O. ( 1 9 7 8 ) Atlas o f P r o t e i n S e q u e n c e a n d S t r u c t u r e , Vol. 5, Suppl. 3, pp. 29--44 M c D o n a l d , C.C., Phillips, W.D. a n d V i n o g r a d o v , S.N. ( 1 9 6 9 ) Biochem. Biophys. Res. C o m m u n . 36, 442--449 Wfithrieh, K. ( 1 9 7 1 ) in P r o b e s o f S t r u c t u r e a n d F u n c t i o n of M a c r o m o l e c u l e s a n d M e m b r a n e s : Probes o f E n z y m e s a n d H e m o p r o t e i n s ( C h a n c e , B., Y o n e t a n i , T. a n d Mildvan, A.S., eds.), Vol. II, pp. 4 6 5 - 4 8 6 , A c a d e m i c Press, N e w Y o r k K r e j c a r e k , G.E., T u r n e r , L. a n d Dus, K. ( 1 9 7 1 ) Biochem. Biophys. Res. C o m m u n . 42, 9 8 3 - - 9 9 1 KeLler, R.M., Wiithrich, K. a n d Peeht, I. ( 1 9 7 6 ) FEBS Lett. 70, 1 8 0 - - 1 8 4 Keller, R.M., Wiithrich, K. a n d Schejter, A. ( 1 9 7 7 ) Biochim. Biophys. A c t a 4 9 1 , 4 0 9 - - 4 1 5 C o o k s o n , D.J., Moore, G.R., Pitt, R.C., Williams, R.J.P., C a m p b e l l , I.D., A m b l e r , R.P., Brusehi, M. a n d LeGall, J. ( 1 9 7 8 ) E u r . J. B i o c h e m . 8 3 , 2 6 1 - - 2 7 5 Keller, R.M. a n d Wilthrich, K. ( 1 9 7 8 ) Biochim. Biophys. A c t a 5 3 3 , 1 9 5 - - 2 0 8 Keller, R.M. a n d W~ithrich, K. ( 1 9 7 8 ) Biochem. Biophys. Res. C o m m u n . 83, 1 1 3 2 - - 1 1 3 9 Senn, H., Keller, R.M. a n d Wiithrich, K. ( 1 9 8 0 ) B i o c h e m . Biophys. Res. C o m m u n . 9 2 , 1 3 6 2 - - 1 3 6 9 Keller, R.M., Wiithrich, K. a n d Schejter, A. ( 1 9 8 0 ) Biochim. Biophys. A c t a 6 2 6 , 1 5 - - 2 2 Dubs, A., Wagner, G. a n d Wllthrich, K. ( 1 9 7 9 ) Biochim. Biophys. A c t a 577, 1 7 7 - - 1 9 4 Wagner, G. a n d Wilthrich, K. ( 1 9 7 9 ) J. Magn. R e s o n a n c e 33, 675---680 B o t h n e r - B y , A.A. a n d Noggle, J.H. ( 1 9 7 9 ) J. A m . C h e m . Soc. 1 0 1 , 5 1 5 2 - - 5 1 5 5 Pettigrew, G.W. ( 1 9 7 4 ) B i o c h e m . J. 1 3 9 , 4 4 9 - - 4 5 9 J e e n e r , J., Meier, B.H., B a c h m a n n , P. a n d Ernst, R . R . ( 1 9 7 9 ) J. Chem. Phys. 71, 4 5 4 6 - - 4 5 5 3 Anil K u m a r , Ernst, R . R . a n d w i l t h r i c h , K. ( 1 9 8 0 ) B i o c h e m . Biophys. Res. C o m m u n . 95, 1--6

320 21 22 23 24 25 26 27 28 29 30 31 32

Keller, R.M. and Wiithrich, K. (1977) Biochim. Biophys. Acta 4 9 1 , 4 1 6 - - 4 2 3 W~thrich, K. (1976) NMR in Biological Research: Peptides and Proteins, North-Holland, A m s t e r d a m Moore, G.R. and Williams, R.J.P. (1980) Eur. J. Biochem. 103, 493--502 Moore, G.R. and Williams, R.J.P. (1980) Eux. J. Biochem. 103, 503--512 Takano, T., Trus, B., Mandel, N., Mandel, G., Kallai, O., Swanson, R. and Dickerson, R.E. (1977) J. Biol. Chem. 2 5 2 , 7 7 6 - - 7 8 5 McDonald, C.C. and Phillips, W.D. (1973) Biochemistry 12, 3170--3186 Moore, G.R. and Williams, R.J.P. (1980) Eur. J. Biochem. 1 0 3 , 5 1 3 - - 5 2 1 Campbell, I.D., Dobson, C.M., Moore, G.R., Perkins, St. J. and Williams, R.J.P. (1976) FEBS Lett. 70, 96--100 Perkins, St. J. (1980) J. Magn. Resonance 3 8 , 2 9 7 - - 3 1 2 Wagner, G., W~thrieh, K. and Tsehesche, H. (1978) Eur. J. Biochem. 86, 67--76 Baumann, R., Anil Kumar, Ernst, R.R. and Wiithrlch, K. (1981) J. Magn. Resonance, in the press Boswell, A.P., Moore, G.R., Williams, R.J.P., Chien, J.C.W. and Dickinson, L . C . J . Inorg. Biochem.

(1980), 13, 347--352