Solution structure of mitochondrial cytochrome c

J. Mol. Bid. (1985) 183,409~428

Solution Structure of Mitochondrial

Cytochrome

I. ‘H Nuclear Magnetic Resonance of Ferricytochrome

c c

Glyn Williams I, Geoffrey R. Moore I, Rod Porteous2, Martin N. Robinson’ Nick Soffe2 and Robert J. P. Williams’ 1 The Inorganic Chemistry Laboratory University of Oxford, South Parks Road Oxford OX1 3&R, U.K. ’ The Department of Biochemistry University of Oxford, South Parks Road Oxford OX1 3&R, U.K. (Received 27 September 1984, and in revised form 18 January 1985) The ‘H nuclear magnetic resonance spectra of tuna and horse ferricytochromes c have been investigated and the resonances of all amino acid methyl groups have been assigned to specific absorption lines. The assignment procedure involves principally the comparison of one-dimensional nuclear magnetic resonance spectra from a range of homologous ferricytochromes c and does not require a prior knowledge of the secondary or tertiary protein structure. Of the 49 methyl groups of tuna cytochrome c, the assignment of 33 is made without reference to the X-ray crystal structure. The method should therefore be applicable to other proteins of similar size where X-ray structures are unavailable. The assignments will be used to investigate the structure of cytochrome c in solution.

1. Introduction Mitochondrial cytochrome c is an electrontransfer protein that transports electrons from cytochrome reductase to cytochrome oxidase in the mitochondrial respiratory chain (Keilin, 1966). It is a small, stable protein (103 to 113 residues; M, 12,000 to 13,000) that can be purified easily, and consequently it has been the object of study with a wide range of techniques in order to define how it performs its biological function (Margoliash & Schejter, 1966; Harbury & Marks, 1973; Lemberg &, Barrett, 1973; Dickerson & Timkovich, 1975; Ferguson-Miller et al., 1979). A prerequisite for a full understanding of the function of cytochrome c is the definition of its structure in the two oxidation stabes, the oxidized state, ferricytochrome c (S = 3) and the reduced state, ferrocytochrome c (S = 0). Chemical and spectroscopic techniques have played a major role in determining the structure of cytochrome c. In particular, the haem attachment residues and axial ligands of the haem were identified as Cysl4, Cysl7, His18 and Met80 in both oxidation states through a combination of chemical 002%2836/85/11040!~-20

$03.00/0

modifications, optical spectroscopy and ‘H n.m.r.7 spectroscopy (Theorell & Akesson, 1941; Margoliash et al., 1961; Harbury et al., 1965; McDonald et al., 1969; Wiithrich, 1969; Redfield & Gupta, 1971; Harbury & Marks, 1973). X-ray crystallographic studies have confirmed and extended these results by the determination of the overall structure at atomic resolution of tuna ferricytochrome c and tuna ferrocytochrome c (Takano & Dickerson, 1980, 1981a,b). Atomic structures of horse ferricytochrome c (Dickerson et al., 1971), bonito ferricytochrome c (Matsuura et aZ., 1979), bonito ferrocytochrome c (Tanaka et al., 1975) and rice ferricytochrome c (Ochi et al., 1983) have been determined by X-ray crystallography, although to lesser resolutions than the strucbures of tuna cytochrome c. t Abbreviations used: n.m.r., nuclear magnetic resonance; n.o.e., nuclear Overhauser enhancement; p.p.m., parts per million; f.i.d., free induction decay; COSY, correlated spectroscopy; EXCTSY, exchange correlated spectroscopy.

409

0 1985 Academic Press Inc. (London) Ltd.

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G. Williams et al.

We are engaged in an investigation of cytochrome c in solution by n.m.r. in order to discover the relationships between its structure and function, and to define the relationships between its amino acid sequence, tertiary structure and dynamic properties. An essential element of such a study is the detailed characterization of the ‘H n.m.r. spectra of cytochrome c, which requires the resolution and assignment of as many resonances as possible. Previous ‘H n.m.r. work with cytochrome c has led to the assignment of most of the haem resonances, many of the aromatic resonances and some of the aliphatic resonances: a total of about 15% of the resonances from hydrogens bonded to carbon (C-H) (Redfield & Gupta, 1971; McDonald & Phillips, 1973; Keller et aZ., 1973; Keller &, Wiithrich, 1978, 1981; Cohen & Hayes, 1974; Moore & Williams, 198Oa,6, 1984; Boswell et al., 1980, 1982; Eley et al., 1982a; Robinson et al., 1983). In this paper, we describe n.m.r. experiments for horse and tuna ferricytochromes c that have led to the assignment of resonances of all their methylcontaining amino acids. These resonances account for an additional 27% of the C-H n.m.r. spectra of horse and tuna ferricytochromes c. The corresponding assignments for ferrocytochrome c are described in the following paper (Moore et al., 1985).

2. Materials and Methods (a) Materials

Horse (type VI), tuna (type XI), pigeon (type XIV), Abccharomyces cerevisiae iso-l (type VIII), and Candidu krusei (type VII) cytochromes c were obtained from the Sigma Chemical Co. (Poole, Dorset, U.K.). Lamprey and

kangaroo cytochromes c were kindly given by Dr R. Wever (University of Amsterdam, The Netherlands). Potassium ferricyanide and sodium dithionite were purchased from BDH Chemicals (Poole, Dorset, U.K.). Oxygen-free argon was obtained from Air Products (Bracknell, Berks. U.K.), ‘H,O (99.8%) from Merck, Sharp and Dohme (Montreal. Que.. Canada), and NaO’H (40% in ‘H,O, isotopic purity 99.0%) and *HCl (35% in *H,O, isotopic purity 99.60/,) from CIBA (Duxford, Cambridge, U.K.). Samples were prepared for n.m.r. by the following procedure: cytochrome c was dissolved in 5 x 10m3 MNaH,PO,/Na,HPO, buffer at pH 7 and a small excess of K,[Fe(CN),] added to ensure complete oxidation. After approx. 30 min, the solution was applied to a column (30 cm x 2 cm) packed with Whatman carboxymethylcellulose (CM-23). previously equilibrated with the phosphate buffer. The column was washed with 150 ml of phosphate buffer to remove inpurities and the ferricytochrome c then eluted with lw-NaCl as a single, sharp band. The eluate was desalted by ultrafiltration and subsequently lyophilized at pH 7. n.m.r. samples of ferricytochrome c were prepared by dissolving weighed amounts of lyophilized cytochrome c in known volumes of *H,O and the pH was adjusted to the required value by the addition of small volumes of concentrated NaO’H and *HCl. Samples of ferrocytochrome c were prepared by the addition of solid sodium dithionite to solutions of ferricytochrome c before final adjustment of the pH. All samples of ferrocytochrome c were prepared in glassware

flushed with Ar. Where required, N-H protons were exchanged for deuterons by heating solutions of ferricytochrome c in *H,O at pH 8 for approx. 2 h at 45°C. Quoted pH values are direct meter readings uncorrected for the small isotope effect (Kalinichenko, 1976). (b) n.m.r. methods n.m.r. spectra were measured at 300 MHz and 470 MHz. The 300 MHz spectra were acquired with a Bruker WH-300 spectrometer equipped with an Aspect 2000 computer system. The 470 MHz spectra were acquired on a spectrometer developed at the University of Oxford, and equipped with an Oxford Instruments Co. superconducting magnet and a Nicolet 1180 computer and 293B pulse controller. Chemical shift values were dioxan standard at measured against an internal 3.741 p.p.m. but all values are quoted downfield of the methyl resonance of sodium-2,2-dimethyl-2-silapentane5sulphonate at 0.00 p.p.m. n.m.r. experiments to obtain Hahn spin-echo and CarrPurcell-Meiboom-Gill spectra, spin-echo doubleresonance, spin-decoupling, saturation-transfer and n.o.e. spectra were carried out in the usual ways (Campbell & Dobson, 1979). In some cases, difference spectra were used to observe small effects. These were recorded for saturation-transfer and n.o.e. spectra in the following way. A pre-saturation pulse of frequency fi was applied before accumulation of each free induction decay. After 8 scans, the f.i.d. was stored, the frequency of the presaturation pulse was changed to f; and a second f.i.d. was accumulated for 8 scans and stored. The frequency was then changed back to f2, the first f.i.d. was recalled into the computer memory, and the cycle was repeated. The value of the frequency fi corresponded to a region of the spectrum where there was no absorption, usually in the and the value of region -1 p.p.m. to -4p.p.m.. frequency f; corresponded to a resonance. Difference spectra were generated by subtraction of the f.i.d. with frequency f; from the f.i.d. with frequency f2. This procedure was adapted for difference spectra resulting from spin-decoupling and spin-echo double-resonance. Resolution enhancement of n.m.r. spectra was achieved as described (Ernst, 1966; Cambell et al.. 1973). Unless otherwise stated. all spectra are resolution-enhanced. The 2-dimensional COSY (Wagner et al.. 1981) and EXCTSY (Boyd et al., 1984) spectra described in this paper and the accompanying paper (Moore et al.. 1985) were obtained at 470 MHz using the published pulse and phase cycling sequences. The time domain data size (tl*t2) was 512*2048 points for COSY and 512*4096 point,s for EXCTSY and the tl dimension was zero filled to improve the spectral definition and allow symmetrization. Resolution enhancement was obtained in both dimensions by convolution with a sine-bell function and the data are presented as absolute value contour plots after double Fourier transformation. (c) Resonance assignment procedure The resonances of interest in this paper are the C-H resonances of methyl-containing amino acids. The assignment procedure falls into 3 stages. The first stage. which involves assignment to a type of C-H proton (e.g. alanine or leucine), is followed by the second stage, which involves assignment to a specific C-H proton (e.g. Ala15 or Thrl9). In many cases a third stage is possible, in which the resonance is correlated with inequivalent protons in the crystal structure (e.g. Leu326, or Leu326,). A complete assignment procedure for the haem

‘H n.m.r.

of Ferricytochrome

411

c. I

Table 1 Characteristics

Amino acid Methionine N-Acetyl Trimethyllysine Alanine Threoninr Isoleucinr

of amino acid methyl ‘H n.m.r. resonances

Number of methyl groups

Primary shifts? (p.p.m.)

1 1 3 1 1

2.13 2.05 3.11 1.40 1.23

2

0.94

0.89 Leucine

2

0.94

Coupled to a group at (p.p.m.)

S S

4.35 4.22

1.89 1.48; 1.19 1.65

0.90 Valine

2

0.97

0.94

Multipletj structure

2.13

li d d t d d d d

t These values are mainly taken from Bundi & Wiithrich (1979). The values for the N-ace@ methyl group and trimethyllysine methyl groups were obtained from spectra of N-acetylmethionine and &im&hyllysine at pH-7 and 25%: - -

$ s, singlet; d, doublet, t, triplet. methyl and thioether methyl resonances of ferricytochrome c has been described by Redfield & Gupta (1971) and Keller & Wiithrich (1978). The experiments are straightforward, involving only saturation-transfer and n.o.e. spectra with specific assignments following from the distribution of substituents of the haem. Assignment of amino acid methyl resonances in proteins is more complex and often the first stage assignment is not clear even with double-resonance experiments (Campbell et al., 1975). The characteristics of amino acid methyl resonances given in Table 1 illustrate why their assignment is difficult; methyl resonances of different amino acids have similar coupling patterns and similar chemical shifts. Furthermore, when the spectrum of a spin system is not first-order, for example in the presence of virtual coupling (Resonance Assignments, section (a)(vii) and Fig. 7), even the multiplet structure may be changed. Pu’evertheless, despite the complexity of n.m.r. spectra containing 49 amino acid methyl resonances (as is the case of horse and tuna cytochromes c), a guide to the first stage assignments can be obtained from the characteristics given in Table 1 by the following procedure. (1) Resolution of the multiplet structure gives direct assignments for isoleucine 6CH, (triplets) and methionine/N-acetyl (singlets) methyl resonances. Spin-decoupling classifies doublet methyl (2) resonances into 3 classes depending upon the differences in chemical shift between the methyl resonance and its coupled resonance. and upon the number of methyl groups coupled to t,he same resonance. These criterra classify the doublet methyl resonances into alanine and threonine resonances. isoleucine yCH, resonances, and leucine and valine resonances. Second stage assignments come mainly from comparison of spectra of related cytochromes c with significantly different amino acid sequences, and from measurements of n.o.e. effects in conjunction with interproton distances obtained from the X-ray structure. The flow diagram in Fig. 1 illustrates the logic of the assignment procedure. The X-ray structure is used as a guide to assignment only as a last resort, and then only from measurements of the inverse 6th power of the distance between protons in conjunction with n.o.e. spectra. Calculations of chemical shift values are not used as a second stage assignment aid. Third stage assignment must of necessity rely on the comparison of n.o.e. data with crystal structure measurements.

3. Resonance assignments (a) Resolved methyl resonances in spectra of tuna ferricytochrome c There are 17, three-proton intensity resonances in the n.m.r. spectrum of tuna ferricytochrome c,

2nd stage osslgnments

Figure 1. Flow diagram showing the logical procedure used for resonance assignment. First-stage assignment identifies a group of coupled spins and attributes them to one of a restricted number of amino acid types, e.g. alanine or threonine, At the level of second-stage assignment, the spin-system is attributed to a specific residue of the amino acid sequence. A subsequent step, third-stage assignment, is required in the case of prochiral centres in order to distinguish chemically equivalent, but magnetically non-equivalent groups within a spin system, e.g. the y1 and y2 methyl groups of a valine residue.

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G. Williams et al

M*lO w9

M*S M*7

M*5

M”6

!,I*4

M*3

M”ll

M*2

H*I

M

I

I

I

I

I

0.4

0.6

I

0.2

I

I

0.0

I

I

/

I

I

-0.4

-0.2 p.p.m.

/

- 0.6

I

1

I

- 2.2

-08

I

I

- 2.4

I

I

I

I

-2.8

-2.6 p.pm.

(0)

M*13 M*12 M*IO M*II

I

0.6

I

I

0.4

I

I

0.2

M*5

I

I

0.0

I

M% MX9 M*6

I

-0.2 p.p.m.

M*4 M*7

i

I

I

-0.4

I

/

-06

I

I

-2.2

- 0.8

(b)

1

1

-2.4

1

I

-2.6 p.p.m.

/

I

I

-2.8

‘H n.m.r. spectrum of (a) tuna and (b) horse Figure 2. The upfield window of the resolution-enhanced ferricytochrome c (27°C. pH* 5.5, 300 MHz). M* denotes an amino acid methyl resonance of the oxidized protein. The peak labelled H*l arises from the thioether methyl group at position 2 on the porphyrin ring. The most upfield methyl resonance (M*l) at approx. -24 p.p.m. has been assigned to MetSO&‘H, and is not shown.

whose secondary shifts are sufficiently large to place them in the upfield window (6 < 0.6 p.p.m.: Fig. 2). and -0.14 p.p.m. The triplets at -0.23 p.p.m. (M*7 and M*9) have been assigned to the 6 methyl groups of Ile57 and Ile85, respectively (Moore & Williams, 1980b; Boswell et al., 1982) and doublets at -2.38p.p.m. (H*l) and -0.43 p.p.m. (M*5) have been assigned to the thioether-2 methyl group (Redfield & Gupta, 1971; Keller & Wiithrich, 1978) and the y1 methyl group of Va128 (Boswell et al., 1982), respectively. The yz methyl group of Va128 is assigned to 0.76 p.p.m. using spin decoupling (see below). A broad resonance at -23.8 p.p.m. (not shown in Fig. 2) has been assigned to the methyl resonance of Met80 (Wiithrich, 1969; Redfield & Gupta, 1971). In accordance with the assignment scheme above, the remaining resonances were first classified into amino acid types using spin-decoupling (Fig. 3). The following pairs of resonances were found to arise from the same CH,-CH-CH, spin system, thus assigning these resonances to the amino acids saline or leucine. They are M*2/M*3, M*4/M*6, M*lO/M*lZ M*ll/M*13. and M*8/M*16, Additionally, the resonances M*5 and M*14 were found to form Val/Leu pairs with methyl Figure 3. Resolution-enhanced difference spectra arising from the spin-decoupling of methyl resonances in the upfield-window region of tuna ferricytochrome c (27”C, pH* 5.5, 300 MHz). The spin decoupling pulse was applied at: (a) 1.90 p.p.m.: (b) 1.50 p.p.m.; (c) 1.20 p.p.m.; (d) l.lOp.p.m.; (e) 0.95p.p.m.; (f) 0+35p.p.m.: (g) 0.75 p.p.m.; (h) 0.70p.p.m.; (i) -0.97 p.p.m.

resonances outside the upfield window, at 0.76 and 0.91 p.p.m., respectively. The remaining resonance M*15 is assigned below to an isoleucine residue.

M*,4jF’ P,p-m 4

(0)

.- -

M*lO MY12 -‘,‘--Jp--------------

(b) M*6 M*4

(c)

[d)

(el

M*3

M*2

‘H n.m.r.

qf Ferricytochrome

413

c. I

HXI

M*9

M*8 M*9

F82 mp

Cd) M*3 L68~

1

I

8

7

I

I

6

I

I

I

5

4

I

I

I

3

I

I

2

I

I

I

0

1

I

-I

I

-2

1

-3

p.p.m.

Figure 4. Resolution-enhanced n.o.e. difference spectra of tuna ferricytochrome c (27”c, pH* 5.5, 300 MHz). The presaturation pulse was applied for 0.5 s at the resonance of: (a) Phe82m; (b) Phe82p+Alal5cc; (c) H*l (haem thioether-2); (d) M*2 (Leu686,). (i) Leucine 68 The upfield shifted resonance M*2 has been observed in all mitochondrial ferricytochromes so far studied, with a chemical shift ranging from c to - 2.66 p.p.m. in horse ferricytochrome -2.78 p.p.m. in tuna ferricytochrome c at 27°C. Such a high degree of conservation, together with the spin decoupling result, which assigns this resonance to a leucine or valine spin system, restricts the assignment of M*2/M*3 to the 6 methyl groups of Leu32 or Leu68. Both Leu32 methyl resonances have been assigned firmly in the diamagnetic reduced protein at -0.61 p.p.m. and -0.51 p,p.m. (Moore & Williams, 1980b; Moore et al., 1985). Saturationtransfer experiments have been used to cross-assign M*2 and M*3 to 0.33 p.p.m. and 1*06p.p.m., respectively, in the reduced protein (Moore et al., 1985) thus restricting their assignment to Leu68. The third stage assignment is made from the n.o.e. between M*2 and H*l (Fig. 4(c) and (d)). A smaller n.o.e. is observed between M*3 and H*l (Fig. 4(c) and Fig. 5(f)). In the crystal structure, Leu686, is 46 A away from the thioether-2 methyl group, while Leu686, is 6.1 A away. Furthermore, a weak n.o.e. effect is observed in the reduced protein between the resonance of Met80s at. -3.25 p.p.m.

and the peak at 0.33 p.p.m. (see Fig. 1 of Boswell et al., 1980). Leu686, is 4.7 A away from MetaO&, while Leu686, is at a distance of over 7.6 8, thus M*2 is assigned to Leu686, and M*3 to Leu686,. (ii) Leucine 35 and leucine 64 Irradiation of the haem methyl-8 resonance at -35 p.p.m. with a one second saturation pulse before acquisition causes n.o.e. effects at resonance M*4, M*ll and M*13 (Fig. 5). Examination of the crystal structure reveals three methyl groups within 4.5 d of haem methyl-& Leu356, (4.1 A), Leu356, (4.4 A) and Leu646, (3.5 A). The Leu646, methyl group is 5.7 d away and the only other methyl group within 6 A is Leu326, (4.9 A). The third stage assignment has been made by observing n.o.e. effects between these methyl groups (Fig. 5). Preirradiation of the resonance comprising M*12, M*13 and M*14 gives a strong n.o.e. effect to the Trp59 C-2 resonance. The only methyl group within 5 A of the Trp59 C-2 proton is Leu356, (3.0 A). This, together with the results of spin-decoupling and pre-irradiation of haem methyl-8 allows the following assignments: Leu356i = M*13, Leu356, = M*ll, Leu646, = M*4, Leu646, = M*6.

G. Williams

et al.

W59C2

M*13

M*4

(dt

ju**

V95a

I

I

I

I

I

I

I

I

I

I

I

I

8

7

6

5

4

3

2

I

0

-I

-2

-3

P.P. m.

Figure 5. n.o.e. difference spectra of tuna ferricytochrome c (WY?, pH* 5.5. 300 MHz). The pre-saturation pulse was applied for 0.5 s at the resonance of: (a) haem methyl+; (b) M*12 +&I*13 +M*14 (Leu986, + Leu356, + Va195yz): (c) M*4 (Leu646,); (d) M*ll (Leu35&); (e) M*9+M*lO (Ile856+Leu986,); (f) M*3 (Leu686,). The assignment of Leu35 is made independent of the X-ray structure by the observation of a triplet resonance at O-2 p.p.m. in the spectrum of kangaroo ferricytochrome c that is linked to haem methyl-8 by an n.o.e., thus implying that a Leu/Val (horse and tuna) to Ile (kangaroo) changeover has occurred. Comparison of the amino acid sequences of horse and kangaroo cytochromes c shows that such a changeover occurs at residue 35 only. The assignment of Leu64 is made independent of the crystal structure by spectral and sequence comparisons with Candida krusei and Neurospora c. There is a three-proton crassa cytochromes singlet at 0.12 p.p.m. in the spectrum of C. krusei ferricytochrome c that is assigned to Met648 by sequence comparisons with N.crassa (M. hT. Robinson, unpublished results). Pre-irradiation of haem methyl-8 gives a strong n.o.e. to this resonance. Comparison of the n.o.e. effect observed in spectra of tuna and horse ferricytochrome c assigns the methyl doublets M*4 and M*6 to the corresponding residue, Leu64. (iii) Leucine 94 Leu94 is a highly conserved residue, present in all the eukaryotic cytochromes studied by us, except that of A;. crassa. Due to the difficulties of comparing n.m.r. spectra from cytochromes with many amino acid changes, assignment is made

using n.0.e. effects and by reference to the crystal structure; rather than by the preferred method of direct sequence comparison. The thioether methyl group at position 2 on the porphyrin ring is within 5 A of three other methyl groups, Leu686, (assigned to M*2), Ile856 (assigned to M*9) and Leu946,. Presaturation of H*l causes n.o.e. effects at M*2 and M*8 (Fig. 4). Effects are also observed at the previously assigned positions of the meta and para resonances of Phe82, both of which are consistent with the crystal structure. The n.o.e. effect at M*2 appears small, probably due to the short T, of this resonance. The spin-related resonances M*8 and M*16 are therefore assigned to the 6, and 6, methyl groups, respectively, of Leu94. Confirmation of this assignment is obtained by pre-saturation of M*2 (Leu686,) when n.o.e. effects are observed at H*l (thioether-2, 4.5 A) M*3 (Leu686,, 3.5 A) and M*8 (Leu946,, 4.7 A: Fig. 4). No other methyl group is within 5.5 A of Leu686, in the crystal structure. (iv) Leucine 98 Like Leu94, Leu98 is a highly conserved haemcontact residue present in all the highly homologous animal cytochromes and replaced by Met98 in only a few of the yeast cytochromes so far studied. Assignment rests on the observation of a weak n.o.e. between M*ll (Leu356,) and M*lO, and a strong n.o.e. between M*ll and a highly coupled

‘H n.m.r.

of Ferricytochrome

peak at 1.49 p.p.m. (Fig. 5). The resonances M*lO and M*12 belong to the same Leu/Val residue and are coupled to a C-H proton at -1.5 p.p.m. Reference to the crystal structure shows that only one Leu/Val y//l C-H proton (other than Leu35y) is within 4.8 A of Leu356,; namely, Leu98y at 3.7 A. The resonances M*lO and M*12 are therefore assigned to the 6 methyl groups of Leu98. The third stage assignment, and confirmation of this result, is complicated by the difficulty of selectively irradiating M*12 (which overlaps M*13 and M*14) and M*lO (which is very close to M*9, Ile856, at all accessible temperatures). However, irradiation of M*9 and M*lO leads to a stronger n.o.e. at M*3 (Leu686,) than can be explained by irradiation of Ile85S alone, which is 7.7 A from Leu686, (Fig. 5). M*lO is therefore tentatively assigned to Leu98d2 (4.5 A from Leu686,) rather than Leu986, (7.1 A from Leu688,). Support for this assignment is provided by the observation of a methionine singlet resonance at 1.04 p.p.m. in the spectrum of C. krusei ferricytochrome c (27”C, pH* 5.3). Apart from the haem ligand Met80, C. krusei has only two such residues, Met98 and Met64. The resonance of Met64 has been assigned to a singlet at 0.12 p.p.m. and the singlet at 1.04 p.p.m. is therefore assigned to Met98. This resonance is 1.09 p.p.m. upfield of its primary resonance position and has a similar temperature dependence to the resonances M*lO and M*12 of tuna ferricytochrome c, which are 1.00 p.p.m. and 0.47 p.p.m. upfield of their primary resonance positions. These observations imply that M*lO/ M*12 and the singlet of Met98 experience similar pseudocontact and diamagnetic shifts and support the assignment of the former to Leu98 (unpublished results).

Of the resonances in the upfield window region of tuna ferricytochrome C, only M*l4 and M*l5 remain to be assigned. Spin decoupling indicates t,hat M*l4 arises from a valine or a leucine residue: CH,-CH-CH, 0.49, 1.9, 0.91 p.p.m. No such spin system is found in horse ferricytochrome c. Furthermore, at 47°C a triplet is resolved Preat -0-5 p.p.m. in the horse spectrum. irradiation of this triplet gives an n.o.e. to a methyl doublet at 0.83 p.p.m. (Fig. 6) that decouples on its own from 1.95 p.p.m. We conclude that these resonances arise from an isoleucine residue that is replaced by leucine or valine in tuna cytochrome c. Sequence comparison indicates that M*14 and the resonance at 0.91 p.p.m. of tuna cytochrome c must therefore be assigned to Va195. The third stage assignment is achieved by pre-irradiation of M*3 (Leu686,) when a strong n.o.e. is observed at 0.49 p,p,m. (Fig. 5). No n.o.e. is expected at the overlapping resonance of Leu3ti1 or Leu986, and the effect must therefore be due to M*l4. The only

415

c. I

methyl group within 4.5 A of Leu686, in the crystal structure is Va195y, (3.6 8) and therefore M*14 is assigned to this group. Va195a is assigned to a resonance at 3-04 p,p.m. This resonance is resolved in n.o.e. difference spectra resulting from the irradiation of Va195yz (Fig. 5) and Leu986, (Fig. 5) and in spin decoupling difference spectra resulting from irradiation of Va195/3. According to the tuna ferricytochrome c crystal structure, Va195cl is 2.7 A from Va195y, and 3.1 A from Leu986,. (vi) Zsoleucine 857, M*15 is coupled to a C-H proton at -0.8 p.p.m. Due to the number of methyl groups in the vicinity of O-8 p.p.m. that are affected by the spin decoupling pulse, it is not possible to assign this resonance to an amino acid type (i.e. Val/Leu or Ile/ Ala/Thr) although, even allowing for pseudocontact shifts, it is unlikely that M*15 arises from alanine or threonine residue, since this would require an upfield shift, of 3.3 p.p.m. at the u/jI proton. The same spin system has been identified in horse ferricytochrome c, where it is possible to obtain a selective n.o.e. from this methyl group (Fig. 6). A peak is obtained in the difference spectrum at - Oa20p.p. m. (Ile85S). Pre-irradiation of He856 gives an n.o,e. at M*15 in both proteins. According to the crystal structure, only four methyl groups are within 6.5 A of Ile856, of which only one is an isoleucine yz methyl group. These groups are the thioether-2 methyl (4.5 A), Thr9y (3.4 A), Ile85y (2.7 A) and Leu946, (4.5 A). Of these, the thioether-2 methyl and Leu946, have been assigned, and Thr9 is absent in horse cytochrome c. M*15 is therefore assigned to the Tle85y methyl group. (vii) ValilLe 28 (threonine 28, h,orse and lamprey) n.o.e. effects are observed on a few aliphatic resonances of horse and tuna ferricytochrome c as a consequence of the irradiation of the haem methyl-5 resonance at - 10 p.p.m. at 27°C (Fig. 6 of Boswell et al., 1982). The CH-CH, spin system of an alanine or threonine of horse ferricytochrome c and the CH,-CH-CH, spin system (M*5/0*76 p.p.m.) of a valine or leucine residue of tuna ferricytochrome c are assigned at the first stage to these affected resonances, as follows: Horse Tuna

CH-CH, 3.07, 0.01 p.p.m. CH,-CH-CH, 0.74, 1.11, -0.46 p.p.m.

The assumption is made that the similar n.o.e. patterns arise because of a substit’ution Val/Leu (tuna) + Ala/Thr (horse) and comparison of the amino acid sequence shows that only two residues satisfy this requirement, These are Va158 (tuna) hThr.58 (horse) and Va128 (tuna) + Thr28 (horse). Of these, the crystal structure shows that residue 28 is close to haem methyl-5 (Va128y,--CH,-5 =-4-O A), while residue 58 is over 14 A

416

G. WilEiams et al.

(d) Horse

47T

(a)

Figure 6. n.o.e. spectra of horse ferricytochrome c (pH* 5.5, 300 MHz). (a) Blank spectrum, 27°C; (b) difference spectrum with pre-saturation for 1.0 s of the resonance of Ile85y,, 27°C; (c) difference spectrum with pre-saturation for I.0 R of the resonance of Ilegy,, 27°C; (d) blank spectrum, 47°C; (e) difference spectrum with pre-saturation for I.0 s of the resonance of Ile956, 47°C.

away. Thus the resonances are assigned to Val/Thr28. n.m.r. studies of lamprey cytochrome c confirm this assignment and remove the dependence on the crystal structure. This protein contains Thr28 and Va158 and it has a similar set of Ala/Thr resonances that are related to haem methyl-5 by 11.0.8. Additionally, this is the only residue that is Ala/Thr in both horse and lamprey and is not conserved in tuna (Table 3). Val28z is assigned to a doublet resonance at 3.11 p.p.m. on the basis of spindecoupling and n.o.e. data (see Fig. 6(c) of Boswell et al., 1982). The unusual appearance of the methyl resonance of Thr28 of horse ferricytochrome c (Fig. 7(a)) is a result of the overlap of its a and p proton resonances. This overlap causes the methyl proton to be effectively coupled to both the a and p protons by the mechanism of virtual coupling (Musher & Corey, 1962). Figure 7(b) illustrates the change in appearance of the simulated spectrum of Thr28 as a fun&ion of the frequency difference between the a and j3 proton resonances (Av). When

Av.= OHa, the simulated and experimental methyl resonances are very similar. The spectral simulation demonstrates how critical is the appearance of the methyl resonance upon Av, a point emphasized by the spectrum of lamprey, ferricytochrome (: (Fig. 7(a)), in which the Thr28 methyl resonance appears as a normal doublet because there are small chemical shift differences between the horse and lamprey spin-systems. (viii) Summary All of the methyl resonances in the upfield window region of tuna ferricytochrome c (6 < O-6 p.p.m.) have now been assigned. One resonance H*l, belongs to a haem thioether methyl and group and it experiences ring current paramagnetic shifts. M*l, the methyl resonance of the iron ligand Met80, is also shifted upfield by the combined effects of ring current and paramagnetic shifts. Another resonance, M*7, is assigned to Ile576 and is present in this region in the spectrum of the reduced protein, being shifted upfield by the combined ring current effects of Trp59 and Tyr74.

‘H n.m.r.

of Ferricytochrome

(0 I

128

417

c. I

well above the haem plane, where a large downfield pseudocontact shift is expected. The assignments are summarized in Tables 2 and 3. (b) Resolved methyl resonances in spectra horse ferricytochrome

T28

I

I

I

I

0.3

0.2

0.1

0

I

I

-04 -0.2 ppm.

-0.3

I

I

I

-05

-06

,,,,,+-Jlie)

I ’ ’ ’ ’ I

0

0.5

3.0

35

I

-0.4

p.p.m.

Figure 7. Spectra showing the appearance of the methyl resonance of Thr28y in (a) horse and (b) lamprey ferricytochromes c. The spectra were obtained at 27°C via Gaussian and have been resolution-enhanced multiplication of the free induction decay. (c) to (e) Simulated spectra of a threonine spin-system (ABX,) with a variety of a-j resonance separations (Av): (c) 0 Hz; (d) 20 Hz; (e) 40 Hz. Note the change in appearance of t,he y-methyl resonance from (e) a doublet with coupling constant 6.3 Hz to (c) a triplet with a coupling constant 3.15 Hz.

The upfield window region of the n.m.r. spectrum of horse ferricytochrome c (Fig. 2) contains 18 three-proton intensity resonances, one more than tuna ferricytochrome c (Fig. 3). Double-resonance experiments, analogous to those described above for tuna cytochrome c, were carried out with similar results. Conserved residues exhibited the same resonance patterns and the substituted residues (Thr28 and Ile95) produced results consistent with the tuna structure. The assignments are summarized in Tables 2 and 3. The assignment of the additional resonance of horse ferricytochrome c is described below. (i) 1soZeucine 9 Carr-Purcell spectra of horse ferricytochrome c at 27°C show three triplets below 0.9 p.p.m. A fourth is evident at higher temperature, and has been assigned to Ile956 (see above). Tuna cytochrome c has only two triplets in this region, which have been assigned to Ile576 and Ile856. Sequence comparison assigns the extra triplet at 0.75 p.p.m. in horse to Ile96. The additional resonance in the upfield window region of the spectrum of horse ferricytochrome c is a three-proton doublet at 0.41 p.p.m. and it is coupled to a C-H proton at 1.65 p.p.m. The resonance pattern observed by spin-decoupling and the chemical shift values indicate t,hat this resonance arises from an isoleucine yZ methyl group. Pre-irradiation at 0.41 p.p.m. (Fig. 6) gives an n.o.e. to the Ile9 triplet as well as to the coupled C-N proton at I.65 p.p.m. The only other isoleucine residues within 14 a of IleSS, using Thr9y of tuna cytochrome c as a guide, are Ile85 and Ile81, both of which are assigned. The peak at 0.41 p.p.m. is therefore assigned to the y,-methyl group of Tle9. This assignment is consistent with the observed n.o.e. effect,s at the resonances of Phe82 (not shown), Ile856 and Leu946,. Additional effects are due to the simultaneous irradiation of He956 (Fig. 6). (c) Methyl resonance8 in the main aliphatic region of ferricytochrome (2

Systematic spin-decoupling in spectra of tuna, horse, lamprey, C. krusei and S. cerevisiae iso-l ferricytochromes

All the remaining resonances arise from haem shifts are whose secondary contact residues attributable to both ring current and pseudocontact effects, the latter being dominant. Only one haem contact residue, Leu32, is missing from this region.

This

residue

containing

is

haem

unique contact

amongst residues

the in being

methylplaced

of

c

c has led to the identification

of

many CH-CH, and CH,-CH-CH, spin-systems belonging to the amino acid types alanine, valine, leucine and isoleucine. threonine, Representative spectra are given in Figure 8. COSY spectra were not helpful for the systematic determination of the first-stage methyl resonance assignments and their complexity was such that they could be interpreted unambiguously only with

G. Williams et al.

418

Table 2 VaZine, leucine and isoleucine spin-systems of ferricytochrome Assignment Valine 3 9 11 20 28 58 66 95 Leucine 32 35 64 68 94 98 Isoleucine 9 57 75 81 85 95

Horse

Tuna B... 2.10. 2.30. 2.45. 1.10. 1.70

Y, 1.06, l”O1 1.31, 1.11 1.14, 0.99 0.76, -0.43 0.79, 0.67

1.90..

0.91,

0.49

y... ? 0.95. 1.29. 0.70. 0.75. 1.50.

6, 1.94, 0.48, -0.33, -0.66, 0.56, 0.45,

6 1.65 0.18 -0.55 -2.78 -0.16 -0.08

B... 1.75. 1.45. 2.20. 0.85.

lk * 3

1’02

2.30. 2.20

1.31, 1.05,

1.10 0.99

6. 1 ‘. ‘. 0.48, -0.22. -0.57, 0.62, 0.48,

1.00: 1.35. 0.70 0.70. 1.50.

y, 6 ..0.79, -0.23 1.29, 1.94 1.13, 0.95 0.53, -0.14 -

Lamprey

2.!0:::

y...

6

-,

y, 1.05, 0.93, 1.36, 1.05.

l&I 0.47 1.10 0.99

1.80. 2.00.

0.79, 1.18,

0.67 1.05

6, ? 1.05: : : 0.48, 1.44. -0.20, 1 -0.56, 1 0.59, 1.50. 0.46.

0.17 -0.55 -2.66 -0.23 -0.11

All chemical shifts measured at 27”C, pH* 5.3. 1, Not determined.

p... 2.10. 165 2.30. 2.20

y...

6 y. 0.41, 0.75 0.83, -0.36 ? 1.26, 1.12. 0.95 0.57, -0.17 0.83, 0.47

p... 1.65. 1.70. 1.45... 2.15. 0.85. 1.95.

c

/?...

y.

1.90. 2.10. 1 1.90’. ‘_‘.

6 0.16 -0.59 -2.67 -0.28 -0.15 6

0.82, -0.34 ? 1.08, 0.92 z -0.25 0196, 2

Residue replaced in this species.

Table 3 Alanine and threonine spin-systems of ferricytochrome Assignment 1 Ala15 Thr78 ThrlQ Ala81 Thr40 Thr49 Thr63 ThrlO2 Ala43 1 1 Thr9 Ala83 Ala23 ? ? 1 1 Ala51 Ala4 ? Ala101 Thr89 ? Ala96 Thr58 ? ! P ‘? Thr47 Thr28

Tuna

Horse

Lamprey

5.92 565 5.53

2.20 3.49 2.22

5.92 5.90 5.56.

2.19 3.46 2.21

5.89 5.53

3.40 2.24

4.85. 4.70. 4.50. 4.50 4.45.

1.29 1.51 1.43 0.87 1.50

4‘90... 1.28 4.70. 1.53 4.60 1.39 4.50 .065 4.65. 1.46

5.10 4.70 4.50. 4.45 4.60

1.32 1.56 1.45 0.85 1.43

4.40 4.30

.0.76 1.28

4.30

1.26

4.20. 4.30

c. krusei 6.30 5.60 562 5.52 5.20

2.31 2.23 3.54 2.23 1.30

4.80. 4.30 4.30, 4.70 4.30.

1.63 1.48

4.10,

1.64

4.00

0.74

490 4.00.

0.77 1.23

3.90

1.26

3.90 3.75

1.26 0.94

4.10,

3.28 2.18 1.32

1.54 1.44

4.65.

1.49

1.39 1.24 1.18

4.40.

1.43

4.30.

1.17

4.30,

1.43

4.20. 4.20 4.15.

1.31 1.22 1.66

4.10. 4.00

1.24 0.75

4.00.

1.51

3.60

1.26

340

1.33

1.23 1.53

1.67

0.70 0.01

3.10

1.79 1.32 1.22 1.67 .0.70

4.00.

1.52

3.40 3.40

1.22 0.83

1.26

-. 3.15. 3.05

4.20. 4.20 4.20 4.10. 4.10.

4.00

S. cerevisiae

5.80 5.55. 5.20

?

4-15. 4.20.

c

0.05

All chemical shifts measured at 27”C, pH* 5.3. For a discussion of the temperature dependence of the ferricytochrome c spectrum, see Moore & Williams (198Od). 1, Not determined. ---, Residue replaced in this species.

419

‘H n.m.r. of Ferricytochrome c. I

T49y (b)

Figure 8. Representative methyl resonance decoupling difference spectra in tuna ferricytochrome c (27°C. pH* 55, 300 MHz). The spin-decoupling pulse was applied at: (a) 3.90 p.p.m.: (b) 4.70 p.p.m.: (c) 2.45 p.p.m.: (d) I.50 p.p.m.; (e) 2.20 p.p.m. the aid of the one-dimensional spin-decoupling data (Williams, 1983). The leucine residues of horse and tuna cytochromes c form a hydrophobic pocket for the haem group, and in spectra of ferricytochrome c their resonances all experience large paramagnetic shifts from the iron atom with the result that all but the resonances of Leu32 are shifted into the upfield window and have been assigned above. Leu32 itself has been assigned using saturationtransfer (see below). Spin-decoupling has been used to resolve the remaining valine p. . . yl, yz and isoleucine B. y methyl spin systems. The complete results, including the assignments of the

isoleucine S methyl resonances are given in Table 2. The decouplings of alanine and theonine residues are readily distinguished from the remainder by virtue of their chemical shift values and coupling patterns (Table 1). The observed a . p and /3. . y decouplings of alanine and threonine residues are shown in Table 3 along with their assignments. (i) Alanine 15 The assignment of Ala15 has been made (Moore & Williams, 198ob) but will be repeated here for clarity. There is a resolved quartet C-N resonance at 5.9 p.p.m. in the spectrum of t.una ferricytochrome c that is coupled to a methyl group at

420

G. Williams

2.2 p.p.m. In the pigeon ferricytochrome c spectrum, the resonance at 5.9 p.p.m. is replaced by a singlet. The only Ala/Thr difference in the sequences of tuna and pigeon cytochromes c that fits this pattern is at residue 15, which is Ala (tuna) and Ser (pigeon). (ii) Alanine IUI Ala15 and Ala101 are the only alanine or threonine residues present in both horse and tuna but absent in lamprey cytochromes c. Ala15 has been assigned (5.9 . . 2.20 p.p.m,) in tuna ferricytochrome c. Comparison of the spin-decoupling data (Table 3) may then be used to assign Ala101 to the decoupling 4-O . . 0.74 p.p.m. in tuna ferricytochrome c. This decoupling is also present in spectra of C. lerusei and S. cerevisiae ferricytochromes c, consistent with its assignment (Table 3). (iii) Alanine 23 (lamprey) Ala23 is uniquely assigned to the decoupling 4.3 . I.53 p.p.m. in lamprey ferricytochrome c, being the only alanine or threonine residue present in lamprey but absent in horse cytochrome c. (iv) Threonine

47 (horse)

The sequences of horse and donkey cytochromes c differ at position 47 only, which is either threonine (horse) or serine (donkey). Consequently, the n.m.r. spectra of these proteins are almost identical. Direct comparison reveals an extra methyl doublet at 0.71 p.p.m. in horse ferricytochrome c (Moore & irradiation at Williams, 198Oc). Time-shared 3.1 p.p.m. decouples this methyl resonance, as well as a one-proton doublet at 3.80 p.p.m. Thus the C&H, /EH and yCH, resonances of Thr47 are assigned to 3.80p.p.m., 3.1 p.p.m. and 0.71 p.p.m., respectively. As expected, this spin system is absent in donkey as well as tuna, horse, lamprey C. krusei and S. cerevisiae iso- 1 ferricytochromes c. (v) Threonine

89 (horse)

Donkey and cow cytochromes differ at two positions; namely, residues 60, Lys (donkey) + Gly (cow) and residue 89, Thr (donkey) + Gly (cow). Their n.m.r. spectra are very similar and direct comparison indicates the presence of an extra in donkey 1.23 p.p.m. methyl doublet at ferricytochrome (57”C, pH* 5.25: Moore & Williams, 198Oc) which is coupled to a C-H proton at 4.0 p.p.m. The analogous spin-system has been ident,ified in horse ferricytochrome c and is assigned to p and y protons of Thr89. This spin-system is absent from spectra of all the other cytochromes studied, which all contain glycine or lysine at position 89. (vi) Threonine 58 (horse) Three alaninelthreonine residues are present in horse, but absent in tuna and lamprey cytochromes c. They are Thr47, Thr58 and Thr89. Of these, Thr47 and Thr89 have been assigned above by comparison with donkey and cow cytochromes c,

et al.

respectively. Thr58 may then be assigned to the remaining unique spin system of horse ferricytochrome c, 3.75 . 0.94 p.p.m. (Table 3). (vii) Threonine 19 and threonine 78 A common feature of the spectra of tuna, horse, C. krusei, S. cerevisiae and rice ferricytochromes c is the presence of a one-proton intensity singlet resonance at -6.3 p.p.m. This resonance is from an clCH proton whose E-J? coupling constant is small. This has been demonstrated, the spectrum being recorded in 90% H20: 10“/6 2H20 when coupling to an N-H proton was observed (Moore & Williams, 1980b). The coupled /?CH resonance at 5.5 p.p.m. was observed using COSY, imploying a 100 millisecond fixed delay at the start of the t, and t, periods to emphasise small couplings (Fig. 9). Timeshared irradiation of the PCH resonance decouples a methyl group at 2.22 p.p.m. thus completing the first stage assignment of these resonances to a CH-CH-CH, spin-system CH-CH-CH, 6.27,5.53,2.22 p.p.m. (tuna ferricytochrome c, 27”C, pH* 5.5). Spin-decoupling has identified CH, spin-system present ferricytochromes.

a second CH-CHin all these

CH-CH-CH, 5.22,5.85,3.49

(tuna ferricytochrome

p.p.m. c, 27°C: pH* 5.5).

Such a high degree of conservation restricts the assignment of these resonances to the a, /I and y protons of Thrl9, He75 or Thr78. The downfield shifts experienced by these residues due to the paramagnetic ferric ion are comparable with the differences in primary chemical shifts between a threonine and an isoleucine amino acid and, for this reason, chemical shift will not be used as a first stage assignment aid in this oxidation state. However, both spin-systems can be cross-assigned in a two-dimensional exchange experiment (Boyd et al., 1984). In the diamagnetic ferrocytochrome c, these spin-systems have chemical shifts characteristic of threonine residues, thus limiting their assignment to Thrl9 and Thr78 (Moore et al., 1985). Secondary assignment is made via chemical modification of the protein using CN-, and is independent of the crystal structure. Previous spectroscopic and chemical studies have established the porphyrin ring is covalently bonded to the protein by thioether links with Cysl4 and Cysl7. and that the side-chains of His18 and Met80 supply the fifth and sixth ligands to the haem iron (Theorell & Akesson, 1941; Harbury et aZ., 1965; Harbury & Marks, 1973). Addition of CN- to ferricytochrome c displaces Met80 and perturbs the spectrum. system n.m.r. The spin 5.22 . 5.85 . 3.49 p.p.m. is markedly perturbed and lost from the downfield spectral region, while the spin-system 6.27 . . . 5.53 . . . 2.22 p.p.m.

‘H n.m.r. of Ferricytochrome c. I

6-

I

I

I

I

I

1 5

6

Figure 9. The aromatic region of a COSY-90 spectrum of tuna ferricytochrome c (46”C, pH* 5.5, 470 MHz). The crosspeak due to coupling between ThrlScr and ThrlSP, for which 3J~/3 < 3 Hz, is emphasized by the inclusion of a fixed 100 ms delay before the evolution period (t,) and before the detection period (tz). A cross-peak due to coupling between the C-Z and C-4 resonances of His26 (3J < 2 Hz) is observed

remains, although shifted slightly from its original position (Fig. 10). The resonances of Ala15 also remain near their positions in the unmodified protein (Z. X. Huang & G. R. Moore, unpublished results), indicating that the displacement of Met80 by CN- does not drastically alter the magnetic properties of the iron or the protein structures above the porphyrin plane, where the bonds at residues 14, 17 and 18 are maintained. Since residues 19 and 78 are constrained by the haem coordination to be above and below the porphyrin plane, respectively, the unaffected spin-system, 6.27 . . . 5.53 . . . 2.22 p.p.m. is assigned to Thrl9, and the perturbed spin-system, 5.22 . . 5.85 . . 3.49 p.p.m. is assigned to Thr78. (viii)

Alanine 83

Alanine 83 has been assigned to the spin-system 4.3 1.28 p.p.m. in tuna ferricytochrome c and 4.3 1.26 p.p.m. in horse ferricytochrome c by a similar procedure to the corresponding assignment for ferrocytochrome c (see Fig. 9 of Moore et al., 1985). This procedure uses an external para-

magnetic probe, [Cr(malonato)3]3-, to broaden the resonances beyond detectability at very low probe concentrations ( < 5%). In human ferricytochrome c, a valine or leucine spin-system is similarly affected (2.10 . . . O-98, 0.95 p.p.m.). The only sequence change that may account for this difference is at position 83, which is valine in human and alanine in horse and tuna cytochromes c. These results are also consistent with the known effects of the probe on the resonances of Phe82 and Tle85 (Eley et al., 19823), and with the absence of t’his spin-system in spectra of C. krusei and S. cerevisiae ferricytochromes c (Table 3). (ix) Alanine

43

Pre-irradiation of the singlet resonance of His26 C-2 in the spectrum of tuna ferricytochrome c gives. an n.o.e. to a methyl resonance at l-50 p.p,m. (Fig. 11). Conversely, pre-irradiation at 1.50 p.p.m. gives an n.o.e. to the resonance of His26 C-2. The crystal structure places Ala438 methyl group at 3.3 A from His26 C-2, and no other methyl group is within 8 A of this histidine C-2.

422

G. Williams et al.

0

Thr I9

Al I

(01

Y

P

k

-

I

(b)

id) Thr 19

I 9

I

11 8

11 7

11 6

1 5

’

1 4

”

1’ 3

” 2

” I

“1 0

-I

1 -2

p.~.lll.

Figure 10. Effects of chemical modification by CN- on the spin-systems of Thrl9 and Thr78 of tuna ferricytochrome c (27”C, pH* 7.3, 300 MHz). (a) Spin-decoupling difference spectrum produced by irradiation of the resonance of ThrlSfl in tuna ferricytochrome c. (b) Blank spectrum of tuna ferricytochrome c showing positions of resonances of Thr78. (c) Blank spectrum of tuna cyanoferricytochrome e. The resonances of Thr78a and Thr78fi are shifted out of the downfield window on addition of CPU’- and have not been assigned in the cyanoferricytochrome c spectrum. (d) Spindecoupling difference spectrum produced by irradiation of the resonance of ThrlSP in tuna cyanoferricytochrome c. Comparison of (a) and (d) shows that only small changes in the chemical shifts of resonances of Thrl9 occur on addition of CN-.

An analogous n.o.e. is observed in the spectrum of horse ferricytochrome c, where the resonance at 1.48 p.p.m. is resolved. In this case, spin-decoupling assigns Ala43a to 4.65 p.p.m. However, in the tuna ferricytochrome c spectrum, the Ala43fi resonance overlaps two other methyl resonances and decoupling occurs from 4.7 p.p.m., 4.45 p.p.m. and 4.1 p.p.m. Combining n.o.e. and spin-decoupling pulse sequences allows the unambiguous assignment of Ala43a to 4.43 p.p.m. in tuna ferricytochrome e (Fig. 11(c)). Th is n.o.e. has been used to resolve Ala43 in spectra of lamprey, C. kruusei and S. cerevisiae ferricytochromes c. (x) Threonine 49 and alanine 51 Two alanine and three threonine residues are present in all five cytochromes studied. Of these Ala43, Thrl9 and Thr78 have been assigned (see above, and Boswell et al. (1982)). The remaining residues, Ala51 and Thr49, must therefore be

assigned to those remaining conserved spinwhich systems, are 4.70 . . 1.51 p.p.m. and 4.15. . 1.63 p.p.m. in the spectrum of tuna ferricytochrome c. The spin-system 4.70 . _ . 1.51 p.p.m. is assigned to Thr49 by observation of an n.o.e. between three 4.17 p.p.m., resonances at 4.70 p.p.m. and 1.51 p,p.m., typical of the effects observed between the a, B, and y protons of a threonine residue (Fig. 11(a), (e) and (f)). The resonance at 4.17 p.p.m. appears to be a singlet under resolution enhancement (Fig. ll), consistent with the predicted a-/3 coupling constant of 3.1 Hz, using a Karplus-type equation (Bystrov, 1976) in conjunction with the crystal structure co-ordinates of Thr49. The spindecoupling observed by irradiating at 4.16 p.p.m. and 1.63 p.p.m. are typical of an alanine residue: no other C-H resonance is observed to decouple, consistent to Ala51.

with

the assignment

of these resonances

423

‘H n.rn.r. oj Ferric~ dochrorne c. I

H26C2

A43 (b)

T49a

(fl -

H26C4

8

7

6

5

4

3

2

I

0

-I

-2

J -3

p.p.m.

Figure 11. n.o.e. difference spectra of tuna ferricytochrome c (27”C, pH* 5.5, 300 MHz). The pre-saturation pulse was applied for 1.0 s at the resonance of (a) A1a4.43/l;S(b) His26 C-2; (c) His26 C-2, wit.h spin-odecoupling at 4.45 p.p.m. during data acquisition; (d) Ile576; (e) Thr498; (f) Thr49a; (g) Ile8lol+Tyr46 o/m; (h) Trp59 C-7.

(xi) Alanine 4 and threonine 9 Ala4 and Thr9 are the only alaninelthreonine residues that are present in tuna, and absent in horse and C. krusei cytochromes c. As expected, two such spin-systems are found that are present only in Of tuna ferrieytochrome spectra c, 4.4. 0.76 p.p.m. and 4.10. . 1.48 p.p.m. lle9 has been assigned in horse ferricytochrome c (Table 2), and the y and 6 methyl groups are found to have a 0.56 p.p.m. and 0.14 p.p.m. shift upfield of their random-coil values, respectively. A similar secondary shift would be expected for Thr9y methyl in tuna ferricytochrome c. The random-coil position of threonine yCH, is 1.23 p.p,m. (Table 1) and thus the methyl group at l-48 p.p.m., if it were Thr9, would experience a 0.25 p.p.m. downfield shift, which is inconsistent with the observed shifts on Ile9 in horse ferricytochrome c. Thr9 is therefore assigned to the spin-system 4.40 . . . O-76 p.p.m. and Ala4 to the spin-system 4.10 . . . 1.48 p.p.m.

(xii) Wzreonine 40, alanine 96 and threonine 102 The only alanine and threonine residues that are present in horse and tuna cytochromes c but absent in C. krusei cytochrome c are Thr40, Ala83. Ala96 and Thr102. Of these, Ala83 may be assigned in both ferricytochrome c and ferrocytochrome c by use of a paramagnetic probe, [Cr(malonato),]3-, in conjunction with sequence comparisons. Three spinsystems that satisfy the sequence data remain in each oxidation state and are listed below. Tuna ferricytochrome c Tuna ferrocytochrome c 4.85 . . . 1-29 p.p.m. 4.75 . . 0.98 p.p,m. 4.50 . . .0.87 p.p,m. 4.50. . .0.87 p.p.m. 3.90. . 1.26 p.p.m. 4.15 . . 1.46 p.p.m. These may be assigned to the resonances of Ala96, Thr40 and Thr102 by a comparison of saturation-transfer, n.o.e. and spin-decoupling data as follows. The spin-system 4,50. . 0.87 p.p.m. of tuna

424

0. Williams

ferrocytochrome c is assigned to Thr40 on the basis of n.o.e. data obtained upon saturation of the resonance of Ile576. Irradiation of the Ile576 resonance in tuna ferricytochrome c produces n.o.e. effects at the resonances of Thr63y and Ile57y, only, contrary to predictions based on the crystal structure (Takano & Dickerson, 1981a,6). This is discussed further by Moore et al. (1985). However, the absence of the spin-system 4.50 . 0.87 p.p.m. during a redox titration under conditions of fast) electron-exchange (see Fig. 7 of Moore et al., 1985) implies that Thr40y has a chemical shift diff’erence between the oxidized and reduced proteins that is greater than 0.05 p.p.m. and therefore precludes its cross-assignment to the spin-system of tuna ferricytochrome c at 4.50. . .0.87 p.p.m. The spin-system 3.90 . I.26 p.p.m. of tuna ferricytochrome c shifts downfield with increasing temperature implying that its pseudocontact shift is negative (Williams et al.: 1985) and that it should the spin system be cross-assigned to 4.15 1.46 p.p.m. of tuna ferrocyt’ochrome c. The spin system 4.85 . . 1.29 p.p.m. shifts upfield with increasing temperature and this, together with the fast-exchange titration data, produces the following assignments and cross-assignments. Residue Thr40 Ala96/Thr102 Thr102/Ala96

Tuna ferricytochrome c 4.85. 1.29p.p.m. 4.50. . .0.87 p.p.m. 3.90 1.26 p.p.m.

Tuna ferrocytochrome c Residue 4.50 .0.87 p.p.m. Thr40 4.75 . .0.98 p.p.m. Ala96/Thr102 4.15 . 1.46 p.p.m. Thr102/Ala96 The cross-assignment of Thr40 to the spin-system c is 4.85 . . . 1.29 p.p.m. of tuna ferricytochrome supported by the observation of an n.o.e. effect between the methyl resonance of this spin system and the resonance of Trp59 C-7 (Fig. 11). The tuna ferricytochrome c crystal structure of Takano bt Dickerson (1981a) places Thr40y 3.6 A from Trp59 C-7, while Ala96fl and Thrl02y are 17.5 A and 12.5 A away, respectively. Additional effects are caused by the simultaneous irradiation of resonances of PhelO (Williams, 1983). However, Ala96fl and ThrlOBy are over 7.8 A away from these protons and would not be expected to receive an n.o.e. from them. of the methyl group at Spin-decoupling 1.26 p.p.m. in tuna ferricytochrome c leads t,o the collapse of a quartet to a singlet at 3.91 p.p.m., characteristic of an alanine c&H proton decoupling. Irradiation of the methyl group at 0.87 p.p.m. causes only small effects at 4.5 p.p.m.> as expected for a threonine fl proton, where a/l coupling is present. Analagous results are obtained for horse cytochrome c, and the following assignments are therefore made. Tuna Tuna ferrocytochrome c Residue ferricytochrome c 0.98 p.p.m. Thr120 4.50. .0*87 p.p.m. 4.75. 4.15. 1.46p.p.m. Ala96 3.90. 1.26p.p.m.

et al. The assignment of Ala96 and ThrlOX, and the cross-assignment’ of their spin-systems between the oxidized and the reduced proteins are confirmed by Moore et al. (1985). (xiii) Threonine 63 and isoleucine ,571; The 8.. y spin-system of Thr63 has been identified in spectra of tuna ferrocytochrome c z!in an n.o.e. effect between the y-methyl resonance of this residue and the &methyl resonance of Tle57. This spin-system is correlated with the decoupling 4.50 1.43 p.p.m. by titrations of ferricytochrome c with ascorbic acid under conditions of fast electron-exchange (see Fig. 7 of Moore et al., 1985); this decoupling is therefore assigned to Thr63. Preirradiation of t’he resonance of Ile576 in tuna ferricytochrome c causes n.o.e. effects at the methyl resonance of Thr63 and a second methyl resonance at 0.79 p.p.m. (Fig. 11(d), and Robinson ef al.. 1983). Spin-decoupling assigns the lat’ter at the first stage to an isoleucine y2 methyl resonance whose PCH proton resonance is at 1.75 p.p.m. Second stage assignment to Tle57y, comes from inspection of the crystal structure, which indicates that Tle57y, is 4.2 A from Ile576, and is the only such methyl group within 8 A. Similar experiments have led to t’he assignment of Thr63 and Ile57y, in horse and lamprey ferricytochromes c. Support for this assignment is provided by the absence of the spin-system 4.50 1.43 p.p.m. in S. cerevisiae ferricytochrome c. where Thr63 is replaced by Asn63. (xiv)

Leucine

32

Roth Leu326 methyl groups have been assigned firmly in the reduced protein using n.o.e. data (Moore et al., 1985) and sequence comparisons, which relied upon replacement of His33 (horse) by Trp33 (tuna) (Moore & Williams, 1980b). Irradiation at the position of Leu326, in the reduced protein leads to weak saturation-transfer effect’s in the mixture, implying that this resonance has a short relaxation time in the oxidized protein. However, irradiation at 1.94p.p.m. in an 80% reduced tuna cytochrome sample leads to partial saturation of the Leu326, resonance of the reduced protein at -0.61 p.p.m. This does not occur in a completely reduced sample and therefore does not arise from an intramolecular n.o.e. The saturation arises as a result of intermolecular electron transfer and thus cross-assigns Leu326, from -0.61 p.p.m. in the reduced protein to 1.94 p.p.m. in the oxidized protein. Leu326, is cross-assigned to 1.65 p.p.m. in tuna ferricytochrome c by a two-dimensional crossexchange method (Moore et al., 1985). (xv) Isoleucine

81

Pre-irradiation of the one-proton doublet at 5.15 p.p.m. in the n.m.r. spectrum of tuna ferricytochrome c gives a strong n.o.e. to a doublet and and methyl group at l-13 p.p.m. a triplet 6.95 p.p.m. (Fig. II(g)). Effects are observed also to coupled peaks at 2.15 p.p.m.. more highly

425

‘H n.m.r. of Ferricytochrome G. I 3.10 p.p.m. and 355 p.p.m. The doublet resonances at 1.13 p.p.m. and 5.15 p.p.m. are decoupled by time-shared irradiation at 2.15 p,p.m. The observed n.o.e. and coupling pattern are characteristic of an isoleucine residue: 5.15. 2.15, 1.13 p.p.m. -C!H-CH-CH,

I

CH, 1.40, 1.60 p.p.m. I CH, 0.95 p.p.m. The position of the YCH resonances have been determined by spin-decoupling. Further n.o.e. experiments show that this residue is present in horse and lamprey but absent in the c. thus restricting C. krusei cytochrome secondary assignment to Ile81 or Ile85. Ile85 has been assigned above and therefore the resonances must arise from Ile81. under consideration Moreover, C. krusei and S. cerevisiae cytochromes possess Ala81 and spin-decoupling has shown the presence of such a spin-system with a similar secondary shift (see Table 3). The use of Carr-Purcell A pulse sequences confirms the presence of the triplet resonance at

0.95 p.p.m. in spectra of horse and tuna ferricytochromes c (Fig. 12) and support its absence in C. krusei ferricytochrome c spectra. (xvi) Isoleucine 75 Systematic-spin decoupling in the region 3.0 to 0.5 p.p.m. in the spectrum of tuna ferricytochrome c has resolved four spin-systems whose decoupling patterns are characteristic of isoleucine /l , . y methyl groups. Three of these spin-systems have been assigned by n.o.e. and sequence comparison to and The fourth, Ile57, Ile8 I Ile85. 1.45. . 1.29 p.p.m., is therefore assigned to the remaining isoleucine residue of tuna cytochrome c, Jle75. The triplet 6 methyl resonance of lle75 has not been resolved clearly in spectra of ferricytochrome spectra shown in c, even in the Carr-Purcell the resonance has been Figure 12. However, assigned at 37°C to a peak at 1.94 p.p.m. in the spectrum of tuna ferricytochrome c by magnetization transfer from the assigned resonance of ferrocytochrome c (see Fig. 2 of Moore et al.. 1985). (xvii) TTaZine 58 Three valinejleucine residues are present in tuna and absent in horse cytochromes c. Of these,

NAc

(a)

1816 A

i

185s 1576

NAc (b)

Figure 12. Multiplet suppression in the ‘H n.m.r. spectrum of ferricytochrome c (27”C, pH* 5.5. 300 -MHz). Triplet and singlet resonances may be resolved selectively by the addition of spectra accumulated using Carr-Purcell A and R pulse sequences with delay times chosen to eliminate doublets with coupling constants of 6.5 Hz (a typical aliphaiic value). Those resonances with long transverse relaxation times give rise to the most intense peaks. The singlet, methyl resonances of the N-acetyl group (NAc) and Met65 (M65) and the triplet methyl resonance of Tle81S are clearly resolved. Ot,her triplet methyl resonances with shorter relaxation times, IleSS, Ile576 and Ile96. may be discerned also. The multiplet structure of Thr28y is discussed in Resonance Assignments, section (a)(vii). The peak labelled with the asterisk arises from an impurity. (a) Tuna ferricytochrome c; (b) horse ferricytochrome c.

426

et al.

G. Williams

(b)

I

IO

I

I

I

I

I

I

I

7

8

9

I

6

I

I

I

I

5

4

I

I

3

1

I

I

I

I

2

I

I

I

J -I

0

p.p.m.

Figure 13. n.o.e. difference spectra of tuna ferricytochrome c (WC, pH* 5.5, 300 MHz). The pre-saturation applied for 0.5 s at the resonance of: (a) PhelO para; (b) Valllcc.

resonances of Va128 and Va195 have been assigned above. Resonances of the third residue, Va158, are assigned to the only remaining spin-system that satisfies this sequence comparison, 1.70 . O-79, 0.67 p.p.m. (xviii) T/‘aZine20 Va120 is resolved in the spectrum of tuna and horse ferrocytochromes c and has been assigned by sequence comparisons (Boswell et al., 1982). Both its methyl groups have been cross-assigned to the spin-system 2.45 . . . 1.14, 0.99 p.p.m. of tuna ferricytochrome c by two-dimensional EXCTSY (Moore et al., 1985). The third stage assignment is achieved by observing an n.o.e. between the methyl resonance at 0.99 p.p.m. and the resonance of PhelO para (Fig. 13). PhelO para is 3.4 A from Val20y, and 4.9 A from Val20y,, hence this resonance arises from ValBOy,. Confirmation of this assignment is obtained from n.o.e. experiments in the reduced protein (Moore et al., 1985). Analogous experiments have been performed with horse cytochrome c and Va120 is assigned to the spin-system 2.20 . . . 1.05 and 0.99 p.p.m. in the oxidized protein at 27°C. The difference in chemical shift of the p proton between these species probably Trp33 (tuna) + His33 arises from the substitution (horse). (xix) Valine 3 and valine 11 At this stage, resonances of only two methyland horse of tuna amino acids containing cytochrome c remain unassigned in the n.m.r. spectra

of the oxidized

Va13 and Valll two unidentified

proteins.

The two residues

must correspond to the remaining spin-systems, which are:

2.30. . 1.31, 1.11 p.p.m. and 2.10. . 1.06, 1.01 p.p.m. for tuna ferricytochrome is present in pigeon

c. The former

spin-system

ferricytochrome c, which contains Ile3 and Valll, thus restricting its assignment to Valll. The latter spin-system,

2.10.

. 1.06,

1.01 p.p.m.,

is therefore

pulse was

assigned

to

Va13. Spin-decoupling difference spectra indicate that a one-proton doublet at 3.86 p.p.m. also decouples upon irradiation at 2.30 p.p.m. Preirradiation at 3.86 p.p.m. causes an n.o.e. effect at a methyl resonance at 1.31 p.p.m., and the resonance at 3.86 p.p.m. is therefore assigned to the EC-H proton

of Valll

(Table

4).

Support for the Valll assignment is given by the observation of an n.o.e. between the UC-N resonance and the resonance of Ala158 (Fig. 13). According to the X-ray structure, Valllcl is 2.8 A from Alal5P, while Val3a is over 8 A distant. (xx) Summary The methyl resonances of all the methylcontaining amino acids of tuna and horse ferricytochromes c have been identified. For tuna cytochrome c, this accounts for 49 resonances from 33 amino acids distributed throughout the primary sequence. The following procedures have been used for second-stage resonance assignment. (1) Comparisons of spin-decoupling data with amino acid sequences, e.g. Thr47 (horse), Ala23 (lamprey).

Table 4 G&H resonances of tuna ferricytochrome Residue Ale4 Vail 1 Ala15 Thrl9 Ala43 Thr49 Ala51 Thr78 Ile81 Ala83 Va195 Ala96 Ala101

6 (p.p.m.) 4.30 3.86 592 6.27 4.45 4.17 4.15 5.22 5.15 4.30 3.04 3.90 4.00

c

427

‘H n.m.r. of Ferricytochrome c. I (2) Chemical modification and sequence comparisons, e.g. Thrl9, Thr78. difference spectroscopy (to (3) Paramagnetic define the nature of the amino acid differences) and sequence comparisons, e.g. Ala83 (tuna), Va183 (human). (4) n.o.e. measurements (to define the nature of acid differences) and sequence amino t’he comparisons, e.g. Va128 (tuna), Thr28 (horse). (5) Correlation of obserSed n,o.e. effects with distances derived from the crystal structure, e.g. Leu98, Leu94. Of these methods, only (5) is dkpendent on the correspondence of the crystal structure and solution structures. are classified In Tables 5 and 6, the assignments dependence on the crystal according to their previous dependence on Their structure.

Table

5

Methyl assignments of tuna ferricytochrome c that are independent of the crystal structure Residue type Ala

Thr

Val

Leu

Ile

Met

Others

15

19

20

35

57

65

NAC

64

1 85

80

1 78

51

+

I+

28

1

49

methyl indicated. The also assignments is resonances of 22 residues have been assigned independently of the crystal structure, while 11 residues have structure-dependent assignments. The assignments are summarized in Ta.bles 2 to 4 and in Figure 13 of the following paper (Moore et aE., 1985). In principle, resonances of all the assigned amino acids may be resolved separately using double resonance techniques or spin-echo pulse sequences, and by taking advantage of their different temperature dependences. Thus many independent probes of the local protein structure are now available. These have been used to define the interaction sites of ferricytochromes c with small redox reagents (Eley et al., 19826; Williams et al., 1982; Moore et al., 1985) and with a biological redox partner of cytochrome c (Eley & Moore, 1983), and in subsequent papers (Williams et al.. 1985; and unpublished results), the assignments are used to define the solution structure of cytochrome c. G.W. thanks Merton College, Oxford for a Senior Scholarship and the Medical Research C!ouncil for a Training Awards fellowship. G.R.M: acknowledges the support of the Science and Engineerifig Research Council through an advanced fellowship and M.N.R. thanks the Medical Research Council for post-graduate support. This is a contribution from the Oxford Enzyme Group.

References

1

81

58

75

9+--95

83 101 61 t 49 etc. indicates that the assignment of residue 51 follows the previous assignment of residue 49. 9 t - 95 indicates that the assignment of residue 9 follows indirectly from the assignment of residue 95 (via Ile95 and Ile9 of horse cytochrome c, see the text). from

Boswell, A. P., Moore, G. R., Williams, R. J. P., Chien, J. C. W. & Dickinson, L. C. (1980). J. Inorg. &o&em. 13, 347-352. Boswell, A. P. Eley, C. G. S., Moore, G. R., Robinson, M. N., Williams, G., Williams, R. J. P.. Neupert, W. J. & Hennig, B. (1982). Eur. J. Rio&em. 124, 289-294. Boyd, J., Moore, G. R. & Williams, G. (1984). J. Mugn. Reson. 58, 511-516. Bundi, A. & Wiithrich, K. (1979). Biopolymers, 18, 285297.

Bystrov,

V.

Alethyl resonance assignments in tuna ferricytochrome c that depend on the X-ray crystal structure Residue type Thr

Val

IkXl

Ile

Met

(1976).

Prog.

NucE.

Magn.

Reson.

Campbell, I. D. & Dobson. C. M. (1979). Mcthon’.sHiochem. And. 25, 1-133. CampbelI; I. D.? Dobson. C. M.. Williams, R. .J. I’. & Xavier, A. V. (1973). J. Magn. Reson. 11, 172-181. Campbell, I. D., Dobson, C. M. & Williams, R. J. P. (1975). Proc. Roy. Sot. ser. A, 345, 23-40. Cohen, J. S. & Hayes, M. B. (1974). J. Biol. Chem. 249,

Table 6

Ala

F.

10, 41-81.

Spectrosc.

Others

5472-5477.

Dickerson, Enzymes

63

R.

E.

& Timkovich,

(Boyer , P. D., ed.),

R. (1975). In The vol. 1I . pp. 397-547,

Academic Press, New York. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L., Cooper, A. & Margoliash, E. (1971). J. Biol. Chem. 246, 1511-1535. Eley, C. G. S. & Moore, G. R. (1983). Biochem. J. 215, II

32 1 68

The assignment of V3 and Vll depends on the prior assignment of all other Leu/Val spin-systems. The assignment is then completed (11 + 3) by comparison with pigeon ferricytochrome c (see the text).

21-21.

Eiey. C. G. S., Moore, G. R.. Williams. R. J. P.. Neupert, W., Boon, P. J., Brinkhof, H. H. K., Nivard, R. J. F. & Tesser, G. I. (1982a). Biochem. J. 205; 153-165. Eley, C. G. S., Moore, G. R., Williams, G. & Williams, R. J. P. (1982b). Eur. J. Biochem. 124, 295-303. Ernst, R. R. (1966). A&an. Mugn. Res. 2, l-135.

G. Williams Fergusson-Miller, S., Brautigan, D. L. & Margoliash, E. (1979). In The Porphyrins (Dolphin, D., ed.). vol. 7. pp. 149-240, Academic Press, New York. Harbury, H. A. & Marks, R. H. L. (1973). In Inorganic Biochemistry (Eichorn, G. L., ed.). vol. 2. pp. 902954, Elsevier, Amsterdam. Harbury, H. A., Cronin, J. R., Fanger, M. W., Hettinger. J. P., Murphy, A. J., Myer, Y. P. & Vinogradov. S. X. (1965). Proc. Nat. Acad. Sci., U.S.A. 59, 165% 1664. Kalinichenko, P. (1976). Stud. Biophys. 58, 235-240. Keilin, D. (1966). The History of Cell Respiration and Cytochrome, Cambridge University Press, Cambridge. Keller, R. M. & Wiithrich, K. (1978). Biochim. Biophys. ilcta, 533, 195-208. Keller, R. M. & Wiithrich, K. (1981). Biochim. Biophys. Acta, 668, 307-320. Keller, R. M. Pettigrew, G. W. & Wiithrich, K. (1973). FEBS Letters, 36, 151-156. Lemberg, R. & Barrett. J. (1973). Cytochromes, Academic Press, New York. Margoliash, E. & Schejter, A. (1966). Advan. Protein Chem. 21, 113-287. Margoliash, E.. Smith, E. L., Kreil. G. & Tuppy. H. (1961). Nature (London), 192, 1125-1127. Matsuura. Y., Hata, T., Yamaguchi, T.. Tanaka, N. 8: Kakudo, M. (1979). J. Biochem. 85, 729-737. McDonald. C. C. & Phillips, W. D. (1973). Biochemistry, 12. 3170-3186. McDonald. C. C., Phillips, W. D. & Vinogradov, 6. h’. (1969). Biochem. Biophys. Res. Commun. 36, 442% 449. Moore, G. R. & Williams, R. J. P. (1980a). Eur. J. Biochem. 103, 493-502. Moore, G. R. & Williams, R. J. P. (19806). Eur. J. Biochem. 103. 503-512. Moore. G. R. & Williams, R. J. P. (198Oc). Eur. J. Biochem. 103, 543-550. Edited

et al. Moore. G. R,. B Williams, R. tJ. I’. (1980d). Eur. ,J. Biochetn. 103. 523-532. Moore. G. R. & Williams, G. (1984). Biochim, Biophys. Acta, 788, 147-150. Moore, G. R.. Eley, C. G. S. & Williams, G. (1984). Adoan. Inorg. Bioinorg, Mech. 3, 1-96. Moore, G. R.. Robinson, M. N., Williams, G. & Williams. R. J. P. (1985). J. i?40Z.Biol. 183,429-446. Musher. J. I. & Cory, E. J. (1962). Tetrahedron, 18, 791809. Ochi. H., Hata, Y., Tanaka, N.. Kakudo. M., Sakurai. T.. Aihara, S. & Morita, Y. (1983). J. Mol. Biol. 166. 4077418. Redfield, A. G. & Gupta, R. K. (1971). Cold Spring Ha,rbor Symp. Quant. Biol. 36, 405-411. Robinson. M. N.. Boswell, A. P.. Huang, Z.-X.. Eley. C. G. S. & Moore, G. R. (1983). Biochem. J. 213. 687700. Takano. T. 8 Dickerson. R. E. (1980). P?oc. ~Vnt. d
by R. Huber

AVote added in proof. Since submission of this and the accompanying papers we have used twodimensional Nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) to obtain resonance assignments which are based solely on the protein sequence, and which are independent of any knowledge of the X-ray structure or the structure of homologous proteins. Of particular importance here is the sequential assignment of resonances of Leu94. Ala96 and Leu98 in both tuna ferricytochrome c and tuna ferrocytochrome c. since these assignments previously relied on predict’ions made using the X-ray structure.