Eur. J. Biochem. 83, 261 -275 (1978)

Structural Homology of Cytochromes c David J. COOKSON, Geoffrey R. MOORE, Robert C. PITT, and Robert J. P. WILLIAMS Inorganic Chemistry Laboratory, University of Oxford Iain D. CAMPBELL Biochemistry Department, University of Oxford Richard P. AMBLER Department of Molecukdr Biology, University of Edinburgh Mireille BRUSCHI and Jean Le GALL Laboratoire de Chimie Bacterienne du Centre National de la Recherche Scientifique, Marseille (Received July 16, 1977)

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c 5 5 2 , the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovihrio vulgaris cytochrome C S S ~ and cytochrome cs54 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.

The question of how cytochromes function as electron-transfer proteins has been discussed many times, but no firmly acceptable mechanism has yet been proposed [l -31. Probably the question can only be answered when the structures of many of these proteins have been determined and their simple physical properties compared within groups of structurally similar proteins. To this end we present a comparative study of structural features of some cytochromes c. Subsequently we shall compare their electron transfer properties. Cytochromes c comprise a class of proteins originally defined through a characteristic absorption at about 550 nm in visible spectra of the reduced protein [4]. This spectral feature arises from a heme c covalently bound to the protein. Furthermore, in all cytochromes c so far investigated, it has been found that covalent bonding is formed by thioether linkages between the heme vinyl substituents and cysteine Ahhrevialion. NMR, nuclear magnetic resonance.

residues on the peptide. In all cases too, a histidine residue adjacent to the second cysteine residue provides one ligand to the iron atom. In spite of these similarities within the cytochrome c class, there is a great diversity of chemical and physical properties. Their molecular weights vary from 7000 to 100000, isoelectric points from pH 3 to pH 11 and redox potentials from 400 to - 400 mV. The sixth ligand to the iron is not defined by the classification and the number of heme groups per polypeptide chain is known to vary from one to more than four [1,5]. Obviously a further division of cytochromes c into closely related subgroups is required. A preliminary sub-classification has been devised on the basis of amino acid sequences since the amino acid sequences of more than 120 cytochromes c are now known [6]. Four types of cytochrome c have been proposed [6]., The largest type (I) has a primary structure similar to that of mitochondrial cytochromesc. A single heme is attached close (10-20 residues) to the N-

+

262

terminus of the peptide. with the sixth iron ligand known or presumed to be provided by a methionine residue about three quarters of the way along the sequence. Type I1 cytochromes c have the hemeattachment site near the C-terminus, and include the higher spin-state cytochrome c'. The third type (111) comprises multiple-heme cytochrome c, and the last (IV) contain5 complex molecules of high molecular weight containing heme c, such as cytochrome c peroxidase. However, even this classification does not really indicate where there are close similarities between the proteins and therefore these types have been further subdivided on the basis of sequence [6]. The type I cytochromes c then fall into the following subclasses : IA. 'long cytochromes c ~ 'mostly , from photosynthetic Rhodospirillaceae; IB, mitochondria1 cytochromes L' and similar proteins from Rhodospirillaceae; IC, 'split-1 cytochromes I" and algal cytochromes,/': ID, cytochromes L' found in denitrifying Pseudomonads (e.g. P. atwginosa ~ 5 5 1 ); IE, cytochromes c5, found in denitrifying and non-denitrifying Pseudomonads, characterized by a second pair of cysteine residues near the C-terminus. Our first aim in this paper will be to outline N M R methods which define the solution structure of cytochromes. We shall then make a comparative study of very closely related proteins. Finally we shall show how it is possible to discern structural similarities between proteins from the different subclasses of type I. In order to use NMR in this way we must choose a standard cytochrome c and clearly define its structure in solution. Here, horse-heart cytochrome c will be the structure on which comparisons are based. The structural deductions will be assisted whenever possible by comparison with known crystal structures. Detailed structures of crystal forms are available for the type I proteins bonito and tuna cytochrome c [7-91, Rhodospi'irillurn ruhrum cytochrome c2 [lo] and Parucocc~usrkmifrificam cytochrome c550 [l I]. Additionally a lo\?.-resolution structure is available for the type I cytochrome ('551 from Pseudomonas aeruginosa [12]. The structures of these type I cytochromes c reveal many common features including not only a common heme co-ordination sphere, but also retained conformational features. Thus it may be suggested that cytochromes c have a common fold. NMR methods provide very suitable data with which to test this hypothesis.

MATERIALS AND METHODS Samples oi' Dctulfbvihrio vulgaris cytochrome c 5 5 3 [13], halotolerant micrococcus cytochrome 1 3 5 4 or 'cytochrome /14 (I)' [14], Rhodospiriflurn ruhrurn cytochrome c2 [I 51, Rhodospiriilum fulvum iso-1 cytochrome c 2 (T G. Meyer and R. G. Bartsch, unpub-

Structural Homology of Cytochromes c

lished results) and Rlzodopseudomoiia,~viridis cytochrome c2 [I 61 were isolated as previously described. Pseudornonus aerugiizosa cytochrome ('551 was provided by Dr J. Melling (Microbiological Research Establishment, Porton Down, Salisbury, SP4 OJG, England) and was purified as previously described [17]. Horse, tuna, rabbit, chicken, pigeon. dog, cow, and Cmdidu krusei cytochromes c were provided by the Sigma Chemical Company (St Louis, Missouri, U.S.A.). Donkey cytochrome c was obtained from Miles Laboratories Ltd (P.O. Box 37, Stoke Poges, Slough SL2 4LY, England). Solutions of all samples were eluted through a 300 x 15-mm column containing Sephadex G-25 (from Pharmacia Fine Chemicals AB. Uppsala, Sweden). to remove low-molecular-weight impurities. They were then lyophilized and dissolved in 300 p1 of 0.1 M deuterated phosphate buffer, at an uncorrected tneter reading of pH 7.0. Mitochondria1 cytochromes L' and Psezrdoornorias aeruginosa cytochrome c 5 5 1 were made up to a concentration of 5 mM; other solutions were made up to the maximum concentration allowed by the available amount of sample, and were all much less than 5 mM. Reduction was carried out using a slight excess of either ascorbic acid or sodium dithionite. Reduced solutions were kept under argon. The NMR spectra were obtained using a Bruker 270-MHz spectrometer operating in the Fourier transform mode, with a magnet from Oxford Instruments Co. Free induction decays were collected in a Nicolet 1085 computer, in which mathematical manipulations were carried out. Convolution difference [181, spindecoupling [19], spin-echo double resonance [20],crosssaturation [21] and nuclear Overhauser enhancement (NOE) experiments [22]were carried out as previously described. Acetone and 1,4-dioxan were used as internal standards but all chemical shifts are quoted in parts per million (ppm) downfield from sodium 2,2dimethyl-2-silapentane-5-sulphonate. ASSIGNMENT : METHODS A N D RESULTS The methods of assigning resonances in an NMR spectrum are well documented [23,24]. The chemical shift position and intensity of a resonance, as well as its multiplet character, are directly observable. The nature of the coupling between multiplets may be determined by the use of time-shared double irradiation, or spin-echo double resonance. These data are often sufficient to make a first-stage assignment. that is of a resonance to a type of amino acid. Second-stage assignment, to a particular residue. is assisted by spectral and sequence comparisons of related proteins. and relevant X-ray data. Second-stage assignment is also often assisted by examination of the the way in which the resonances have been perturbed from their posi-

263

D. J. Cookson, G. R. Moore, R. C. Pitt, R. J. P. Williams, 1. D. Campbell, R . P. Ambler, M. Bruschi, and J. Le Gall Table 1. Properties ojc:,,tochromes c I n the aromatic amino acid composition, W Protein

=

tryptophan, H

Redox potential

=

histidine. F

Molecular weight

=

phenylalanine, Y

Type

=

tyrosine. n.d.

=

not determined

Aromatic ammo acid composition ~~~~

Reference

~

W

H

F

Y

IA IB 1B IB IB IB IC

1 I 2 1 1 0 0

2 3 2 4 2 2 1

4 4 3 4 6 2 0

5 4 5 5 3 5 5

IC ID IE

2 2 0

3 1 1

1 2 0

5 1 6

mV Rhodaspirillum ruhrum c2 Horse c Tuna 1’ Candida krusei c Rkodupseudonionus viridis c2 Rhodospirillun? firhum iso-I c2 Halotolerant micrococcus c544 Euglena gracilis (‘552 Pseudonzona.s aeruginosa c55 Desufphovihrio vulguris c553

+ 320 + 255 + 265 + 260 n.d. n.d. + 113

+ 360 + 286 0 to -60

13000 13500 13500 13000 13000 13000 1x000 (2 x 9000) 9 000 8 100 9 000

* Ambler, R. P., Meyer, T. E., and Bartsch, R. G., unpublished results. It is not clear to which group this protein belongs, it has been tentatively classified as type IE.

tions in the free amino acid, and of the effects of changing temperature, p1-I and oxidation state. In this section we present a brief account of the first and second stage assignments of the resonances in spectra of horse cytochrome c, followed by a description of similar resonances that have been observed in spectra of some other mitochondria1 and bacterial cytochromes c. This procedure is adopted because the NMR spectra of horse cytochrome c have been more thoroughly investigated than any other cytochrome spectrum (Moore and Williams, unpublished results). The proteins we shall discuss are shown in Table 1. Assignment of resonances in spectra of the bacterial cytochromes c relies partly on spectral comparisons with horse cytochrome c, but independent assignments based on comparison between spectra of the bacterial cytochromes c are also made. Second-stage assignment is generally difficult, and is more reliable when all the resonances of a particular type of residue have been identified. For horse cytochrome c assignments have been made for the sole tryptophan residue, the four phenylalanine residues and two of the four tyrosine residues. Similarly resonances of all the aromatic amino acids of Pseudomonas aeruginosa cytochrome c 5 5 1 , Desulfovibrio vulgaris cytochrome c553 and halo-tolerant micrococcus cytochrome c554 have been assigned. However, for the cytochromes c2 from Rhodospirillaceae the greater aromatic amino-acid content, coupled with a limited supply of protein, have prevented a complete firststage assignment. MITOCHONDRIAL CYTOCHROMES c

Fig. 1 shows the aromatic region of the convolution difference spectra of horse heart ferrocytochrome c,

1

t

I

I

I

I

I

10

9

8

7

6

6 (PPW

Fig. 1. Aromatic region of ihe convolution difference .spectra of ( a ) korse.ferroc~tochromec, ( b ) tuna.f~,rrocytochromec and ( c ) Candida krusei .fbrrocytochrornec. Samples were 5 mM in ’H20 at 57 ‘C, pH = 5.5. N H protons have been exchanged in (b)

Candida krusei ferrocytochrome c and tuna ferrocytochrome c. Horse Cytochrome c

The assignment of the resonances of horse cytochrome c has been carried out in detail. The heme group resonances and resonances arising from 18 of the 104 amino-acid residues have been assigned at the second-stage level [35 -431. The second-stage assignments were established by using a combination of spectral and sequence comparisons between homologous proteins, and by the use of the effects of extrinsic and intrinsic probes on the chemical shifts and linewidths of the resonances

264

Structural Homology of Cytochromea c

Table 2. Chernic t i / s h i f r s o/ the liistirline und methionine Iigand resonances in Ferroe.vrocliromrs Conditions of the protein arc as in Fig. 1 and 6 Protein

HIS C-4

Met j - C H

Met 1;-CH

Iiorse c C'. krusei c' P. neru~inoscic , ~ ~

0.13 0.11 0.15

- 0.19

-

~~~~~~~~

n.d. -- 0.52

Rps. viridis ('L R.,/ulvum iso-1 c2 R. rubrum1 ( ' 2 Halotolerant micrococcus

1'554

n.d. n.d. 0.09 0.06 0.25

0.3 n.d. n.d. n.d. - 0.90 -

~~

1.87 - 1.78 0.87 -

1.2 1.89 1.91 - 1.23 3.20

2.58

- 2.43 -

Met CH1

Reference

--

3.73 3.71 3.52

- 3.28

[35.36]. this work this work 117.451

- 3.13

-

3.1

-

-

-

- 3.26

-

-

-

-

- 2.71

-

-

2.67

- 2.77 -

-

2.72

-

-

Met 1;-CH

~~~~~~

-

w

I

tgrucilis csjz

Met /(-CH

2.43

3.1 3.77 3.80 - 3.52 - 4.04

- 2.90

2.76 3.16

- 2.85 -

3.52

[441 this work this work [50,51], this work this work

7 - 0.67

~

1.76

in conjunction with the crystal structure of tuna cytochrome c (see later). Note that the resonance assignments for only six amino-acid residues rely upon data obtained from the X-ray structure. The tnethods of resonance assignments have already been reported [35-431. The assignments of Trp-59, Phe-82, Tyr-48, and Phe-46 were given at a conference in 1976 [43], those of His-18 and Met-80 are well established [35,36]. We will not repeat the assignment procedure here but a number of features need to be stressed as they will be used several times in this paper in order to assign resonances of other cytochromes. His-18 and .Met-80. It has been well established that the 5th and 6th iron ligands of horse cytochrome c are His-18 and Met-80 [35,36]. The proximity of the axial iron ligands to the heme causes the resonances of the ligand nuclei to experience large upfield chemical shifts, and in spectra of the diamagnetic reduced protein these were readily identified, as in most cases these shifts are sufficient to remove the resonances

-

3.43

- 3.87

- 3.63

this work

from the main protein envelope (Fig. 2). Table 2 records the chemical shifts of resonances assigned to the histidine and methionine ligands. Positive assignment of the Met-80 /I-CH resonance at - 0.19 ppm was obtained by observation of a nuclear Overhauser enhancement from the previously assigned /?-CH resonance at --2.58 ppm. Trp-59. Fig. 3 shows the cross-assignment of the Trp-59 resonances of horse ferricytochrome c and ferrocytochrome L'. The large shifts experienced by these resonances arise through ring-current and pseudo-contact shifts from the heme group. In the spectrum of ferrocytochrome c the tryptophan resonances experience only a ring-current shift from the heme whilst in the spectrum of ferricytochrome c they experience an additional pseudocontact shift. For a tryptophan residue far from the heme (for example. Trp-33 of tuna cytochrome c) there is little or no contribution to the resonance chemical shift from the heme ring-current or pseudo-contact shift mechanisms in either oxidation state. Thus change in oxidation

D. J. Cookson, G. R. Moore, R. C. Pitt, R. J. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, and J. Le Gal]

8

5

6

7

265

6 (ppm)

Fig. 3. The assignment of tryptophan resonances. (a) L-Tryptophan, (b) Trp-59 of horse ferricytochrome c and (c) Trp-59 of horse ferrocytochrome c. Dashed lines show resonances connected by spin decoupling, full arrows show resonances connected by cross assignment in a 50 7" mixture of ferricytochrome c and ferrocytochrome c Table 3. Chemical shifts and coupling patterns of the benzenoid resonances of mulogous tryptophan residues of .ferrocytochrome c Conditions of the protein are as in Fig. 1 and 6 or as in references quoted. Id = one proton doublet (C-4 or C-7); 't = one proton triplet (C-5 or C-6) Sequence position

Protein

Horse c Horse c Horse c Tuna c C. krusei c P.aeruginosa c 5 5 1 E . gracilis c552 Rps. viridis c2 R. rubrum c2 a

(FeIII) (COIII) (Fell) (FeIT) (FeII) (FeII) (FeII) (FeII) (FeII)

59 59 59 59 59 56 59 58 62

Shifts

Reference

'd

't

't

Id

7.37 7.59 7.60 7.60 7.18 7.36 7.54 7.54

6.54 6.62 6.68 6.64 6.82 6.38 6.35 6.78

6.31 5.77 5.74 5.76 5.65 5.81 5.48 5.58 5.88

7.57 7.07 7.07 7.07 7.28 7.10 6.87 7.28

-

~

[41,43l [461 142,431 ~421 this work 1171 1471, this work this work this work

-

Resonances not identified, see text.

state does not produce a change in resonance chemical shift position. This example makes it clear how it is possible to distinguish residues close to and far from the heme. The proximity of Trp-59 to the heme was established further from the temperature dependence of the tryptophan resonances in the spectrum of ferricytochrome c and from assignment of the Trp-59 resonances in the spectrum of horse cobalticytochrome c [46]. The chemical shift positions and coupling pattern of the benzenoid resonances of Trp-59 are shown in Table 3. Phe-82. Fig. 4 is a schematic diagram outlining the assigned resonances of this phenylalanine residue, and their chemical shift positions are tabulated in Table 4. This coupling pattern identifies the resonances as belonging to a phenylalanine residue, and the lack

Table 4. Chemical shifts and coupling patterns of the resonances of analogous phenylalanine residues o jferrocytochrome c Conditions as for Fig. 1 and 6 except horse ferrocytochrome c at 97 "C; indicates resonances not observed (see text) ~

Protein

SeShifts of protons quence POortho meta para sition ~~~~

References

PPm ~~~~~~~

Horse c Tuna c C . krusei c R . rubrum c2 Rps. viridis c2 R. fulvum iso-l

c2

82 82 82 93 81 79

7.10 -

~

6.70 ~

-

-

-

-

7.28 7.27

7.09 7.03

6.34 6.33 6.16 6.16 6.50 6.48

[43] [43] this work thiswork thiswork this work

Structural Homology of Cytoclirornes c

266

Ferricytochrome c

Ferrocytochrorne c

-c

37OC

c H R

F'he - 46

~

1 1 1 15OC /

\ \

\

\

\

I

\ \ i //

Tyr 48

\

/

\

/

'

\

\

/

/

\

/

I

x'

/

/

.LA 97OC

Table 5. C ' h m i r t ill d i i f t x and c~iuplingpatterns of the re~onance.~ (if crnalojious f ~ r o , \ l ~re.\idul~,s lc ~lf:fc,.i.oc.~~toehror?lr c Conditions as for Fig. I for bacterial proteins; horse ferrocytochrome c. a t 97 c'. tuna ferrocytochrome c at 77 C. 'd -2-proton doublel. not unambiguously assigned to orrho and n~cruresonances Protein

Sequence Chemical shift . position 'd 'd ~

~~~~

Reference

PP'n

Horse 1, Tuna c /$I,\. lJ/r;d;\ ( ' 2 R. riihriwi c

K. f i r l w r r i i iso- 1 < 2

48 48 47 48 43

6.78 6.78 6.87 6.76 6.59

6.18 6.18 6.22 6.04 5.82

121,431 [21,431 this work this work this work

of observable resonances for the orfho and J W ~ W protons at temperatures below 87'C indicates that the residue undergoes slow flipping, causing exchangebroadening [43] of the resonances arising from the ortho and meta protons. In the spectrum of ferrocytochrome c, the upfield ring-current shifts in the order puru > meta > ortlzo place this residue in the cone of a ring-current [48]. The large downfield shift experienced by the para proton resonance in the spectrum of ferricytochrome c and its temperature dependence demonstrates that this ring-current centre is the heme group [43]. From the X-ray structure only Phe-82 is in a position for its resonances to possess these properties. Tyr-48 and Phe-46. The temperature dependencies (see Fig. 5) of the resonances of Tyr-48 and Phe-46

D. J. Cookson, G. R . Moore, R. C. Pitt, R. J. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, and J. Le Gall

revealed that these residues are flipping about their C-F-C-y axis at a rate of 320 s-' at 57"C, with an activation enthalpy which is independent of temperature over the range 0-97 "C [21,43]. The analogous resonances in rabbit, beef, chicken, donkey and tuna ferrocytochrome c show a very similar temperature dependence. The chemical shift positions of the resonances of Tyr-48 for horse and tuna ferrocytochromes c are shown in Table 5. In the slow exchange region, at lower temperatures, a tyrosine residue gives rise to four one-proton doublets, while in the fast exchange region, at higher temperatures, two two-proton doublets are observed. At intermediate temperatures the peaks broaden and disappear. A similar process is observed for the ortho and meta proton resonances of a phenylalanine residue. The para proton triplet is unaffected throughout the transition. In favourable circumstances these changes are readily observed in an NMR spectrum [21]. Thr-78 or Thioether Residues. The one-proton quartet at 6.36 ppm in the spectrum of horse ferrocytochrome c is coupled to a three-proton doublet at 2.57 ppm. This coupling pattern can only arise from a CH-CH3 group, that is from an alanine or threonine [49] residue, or from the thioether heme substituent. The application of a gated irradiation pulse at 6.36 ppm causes a reduction in intensity of the heme meso proton resonance at 9.65 ppm. This negative nuclear Overhauser enhancement coupled with the large downfield shift from their primary positions indicates that the resonances at 6.36 and 2.57 ppm arise from an alanine or threonine or a thioether residue very close to, and in the plane of, the heme. Horse cytochrome c contains six alanine residues but only two of these are close to the heme [8]. Sequence and spectral comparisons with pigeon and Candida krusei cytochromes c eliminate these alanine residues as being responsible for the above NMR signals. The only threonine residue shown by the X-ray structure of tuna cytochrome c to be near enough to the heme for the observed nuclear Overhauser enhancement is Thr-78. Moreover, Thr-78 is conserved in all mitochondrial cytochromes c. On the basis of these facts and an earlier assignment of the two thioether groups [35] we assigned the resonances at 6.36 and 2.57 ppm to Thr-78. The assignments are not final, however, since Wuthrich and Keller disagree with the latter assignment and two alternatives are given in Table 6. Another one-protein peak at 5.2 ppm also gives a negative nuclear Overhauser enhancement to a heme meso proton resonance at 9.38 ppm and this has been recently assigned' to a thioether proton. The Protein Fold. These and other chemical shift positions of assigned resonances in the spectra of horse

'

We thank Prof. K. Wuthrich for supplying the information after this paper was submitted.

267

Table 6. Chemical shifts and coupling patterns q f rcsonances of uliplzatic g r o ~ i p near s the heme Conditions as for Fig. 1 and 6 or as in reference quoted. NOE = nuclear Overhauser enhancement. Aliphatic resonance = oneproton peak, often quartet resonance. 3d -3-proton doublet. I n the assignments sequence; numbers refer to horse cytochrome c Protein

Shifts

Assign- Referment ence NOE coupled ahphatic resonance

methyl resonance 3d

9.65 9.38 9.65 9.38 9.68 9.36 9.34

6.36 5.2 6.34 5.2 6.35 5.2 6.16

2.57

9.86 ?

5.98 6.0

1.85 ?

9.73 8.92 9.45 9.23 9.31 8.60 9.50 9.16

6.21 4.95 6.32 5.69 6.01 5.29 5.73 5.26

1.88 2.39 2.41 2.37 2.26 ?" ?"

9.60 9.18

5.75 6.12

2.20 2.39

meso

resonance

PPm Horse c Tuna c

C. krusei c P. aeruginosa

-

2.56 -

2.55 -

2.41

cs5 1

E. gracilis

('552

Rps. viriclis c2 R . rubrum c2

R. fulvum iso-1 c2 Halotolerant micrococcus

Thr-78h [43] this work Thr-78 this work this work Thr-7Sh [43] this work

-

this work this work

[451, this work this work this work this work this work this work this work this work this work

c554

D. vulgaris cs53

a

this work this work

Structured resonance not seen clearly. These resonances could be part of a thioether group

ferrocytochrome c and ferricytochrome c provide evidence about the relative positions in space of the nuclei concerned with respect to the heme group. Additionally, the thioether ligands fix the positions in space of the Cys-Ala-Gln-Cys sequence providing a total of 22 amino-acid residues, well spaced out along the length of the protein (Table 7) whose position relative to the heme group of horse cytochrome c in aqueous solution are known. In the light of this knowledge we can state with a high degree of confidence that the fold of horse cytochrome e in solution is the same as that of cytochrome c in the crystal.

Other Mitochondria1 Cytochromes c There is an almost exact correspondence between the perturbations of the assigned resonances of between 14 and 18 amino-acid residues for the cyto-

Structural Homology of Cytochromes c

268 Table 7. Resonuncc~u.ssignmerits in h e .spectra of horse cytoclirome c Groups printed in bold-face type are those for which an assignment has been made

'

Ac-GI y -Aap-Vnl-Glu-Lq s-G ly-Lys-Lys-Ile-Phe'"-Val-Gln-Lys-Cys-Ala-Gln-Cys-His-Tlir-Valz0-Glu-Lys-Gly-Gly-Ly~-~is-LysL heme -7 hr- -Phe-Thr-Tyr-Thr-Asp5'Ala-Asn-Lys-AsnLy\-Gly-Ile- Ihi Trp-LL\"G-CJlu-Glu-Thi -Leu-Met-Glu-Tyr-Leu-Glu-Asn-()-Pro-L>\-L !\-Tyr-Ilc-Pro-Gly-Thr"-Lys-~et8"-11e-Phe-Ala-GIj-l le-Lq ~-Ly~-L~~-Thr-Glu'"-Arg-Glu-Asp-Leu-Ile-AIa-Tyr-Leu-L b s-L\ s"""-~la-Thr-A\~i-Glu * Assignment doubtful (see tcxt).

chromec from tuna, donkey, rabbit, dog, cow and Candida kru.wi to those of horse heart cytochrome c. Therefore these observations permit the conclusion that all these closely related class IB cytochromes c have the same protein fold. In addition t o the aminoacid assignments given in Table 7 we have further assignments for some of these mitochondria1 proteins, for example His-39 [40] and AT-trimethyl-lysine-72 of Candida krusei cytochrome c, which confirm the conformational si niilarit y. BACTERIAL PROTEINS

We shall proceed with the comparative study of other cytochromes c as follows. We establish first that all the cytochromes c under discussion have the sulphur atom of a niethionine residue as the sixth iron ligand. Then, from the knowledge of the Cys-Xxx-Xxx-CysHis peptide which binds the heme to the protein, we know that all have a common metal-coordination sphere. We then examine the aromatic region of the NMR spectrum in order to see if each cytochrome has a similar heme environment as far as aromatic amino acids are concerned. Resonances from groups near the heme arc perturbed in an easily characterised manner. The next group of amino acids which we can place in space close to the heme are those which have resonances which can be shown by nuclear Overhauser experiments to lie close to protons of the heme. These are some of the aliphatic amino-acid side chains. Elsewhere in the cytochrome fold we can look for close proximity of groups to one another through mutual spectral perturbations e.g. ring-current shifts which are not due to the heme group. (Note that in this paper we shall not explore the use of externally added probes so as to describe the protein surface but this will be done subsequently.) On the basis of some 20 observations on some 10 amino acids dispersed through the sequence we are then able to make statements a s t o the relationship of the fold of a protein to that of horse heart cytochrome c. Fig. 6 shows the aromatic regions of the convolution ditrerence spectra of Pseudomonas aeruginosa

Fig. 6. The aromatic, region of' the c~orivolutioncl(ff&ww s p c ' t r u of' various cytocliromes c. (a) Pscadomonas aerugino.~trferrocytochrome ~ 5 5 1 (b) , R/iodo.spiri//umJulvzrm iso-1 ferrocytochrome c ~(c) , Rhodopsrudoinonas viridis ferrocytochrome ('2, (d) Rhodospirillum rirbrum ferrocytochrome c2, (e) halotolerant micrococcus ferrocytochrome ('554 and (f) Desulfovibrio vulgaris ferrocytochrome ~ 5 5 3 Sample . (a) was 5 mM, the others were made up to the maximum available concenlration, always less than 5 m M . Solutions were 0.1 M in deuterated phosphate buffer at pH 7.0 and 57 C

ferrocytochrome c5s1, Rhodospirillzim rubrum ferrocytochrome c2, Rhodopseudoomonus vfridis ferrocytochrome c2, Rhodospirillum julvum iso- 1 ferrocytochrome c2, Desulfovibrio vulgaris ferrocytochrome c553 and ferrocytochrome c5s4 from a halotolerant micrococcus. All the spectra contain sharp, well-resolved resonances. The number of peaks in the spectra vary considerably, reflecting the differences in aromatic amino-acid composition (Table 1). The first-stage assignments, to types of amino acid, were carried out using the methods of double resonance previously described [18,19]. The N M R spectra of Pseudomonas aeruginosa cytochrome ('551 have already been described in some detail [17]. Some studies of the heme ligand resonances of ferrocytochromes c2 have been published [50,51]; but this is the first detailed study of their aromatic resonances. The spectra of halotolerant micrococcus ferrocytochrome ('554 and Desulfovibrio vulgaris ferrocytochroine c j 5 3 are reported here for the first time. Chemical shift data and

D. J. Cookson, G. R. Moore, R. C. Pitt, R. J. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, and J. Le Gall

coupling patterns for Euglena gracilis ferrocytochrome c552 were taken from the literature [44,47]. We turn now to individual resonances in the spectra, especially to those arising from groups near the heme. Assignment of the Resonances of the 5th and 6th Iron Ligands

Knowing the general pattern and position of resonances expected to arise from the histidine and methionine iron ligands from a study of mitochondrial cytochromes c, enabled rapid confirmation that all the proteins under study contain a methionine ligand and most contain a histidine ligand. Table 2 shows the chemical shift positions of resonances assigned to the histidine and methionine ligands of these proteins. Additional assignments are included in some cases for the histidine C-4 resonance. Where the histidine resonances were not observed sequence parallels permit the conclusion that there is a histidine ligand. Assignment of the Pseudomonas aeruginosa Met-61 P-CH and y-CH resonance come from nuclear Overhauser experiments and from exchange characteristics of the resonances in mixtures of oxidised and reduced protein. Now that it is known that all the proteins have the same coordination sphere we can assume that in the reduced state the pertubations of the resonances of group close to the heme but not bound to the iron will be virtually the same from one protein to the next provided there are identical amino-acid side chains in very similar positions in space close to the heme. Assignment o j Tryptophan Resonances

Tryptophan resonances were identified by the process outlined for horse cytochrome c. In every case except Rhodospirillum rubrum cytochrome c2 all expected benzenoid resonances were observed. As may be seen from Table 1, the second-stage assignment of tryptophan resonances to specific tryptophan residues is trivial for the cytochromes from Rhodospirillum rubrum 1251 and Rhodopseudomonas viridis (Ambler, R. P., Meyer, T. E., and Bartsch, R. G., unpublished results) which only contain one tryptophan residue apiece. Pseudomonas aeruginosa cytochrome c551 contains two tryptophan residues [32]. The second-stage assignment in this case was made on the basis of sequence comparisons and spectral properties [17]. One set of tryptophan resonances has a strong dependence on both temperature and oxidation state, and their chemical shifts positions in the spectrum of ferrocytochrome c551 at 57 "C are closely similar to those of the only tryptophan residue in the above proteins and in horse-heart cytochrome c. These reso-

269

nances are therefore assigned to Trp-56, which has been aligned with Trp-59 of horse cytochrome c. The other set of resonances have the properties of a residue far from the heme, and are assigned to the other tryptophan residue, Trp-77 [17]. Table 3 shows the chemical shift positions and second-stage assignment of the tryptophan resonances for all tryptophan-containing ferrocytochromes c. The choice of which set of tryptophan resonances for Euglena gracilis cytochrome c552 to include was made on the basis of chemical shift positions. The secondstage assignment of these resonances to Trp-59 of Euglena gracilis cytochrome c552 is a suggestion based upon sequence comparisons. Insufficient sample of Rhodospirillum rubrum cytochrome c2 was available to achieve adequate signal-to-noise ratios for doubleresonance experiments to confirm the assignment of the one-proton triplet at 5.88 ppm.

Assignment of Phenylalanine Resonances

The first stage of assignment of phenylalanine resonances was carried out as described for horse cytochrome c. The second-stage assignment of these resonances, to specific phenylalanine residues, was based upon spectral and sequence comparisons, in conjunction with the X-ray structure of Rhodospirillum rubrum cytochrome c2 and will be reported fully elsewhere. Here we concentrate on the assignment of resonances to the phenylalanine residues analogous to Phe-82 of mitochondrial cytochromes c, which are conserved in bacterial cytochromes c2 (Ambler, R. P., Meyer T. E., and Bartsch, R. G., unpublished results). The expected proton resonances of both phenylalanine residues of Rhodospirillum fulvum iso-I ferrocytochrome c2 have been identified. Those shown in Table 4 have the same pattern of shift from their primary positions as those of horse cytochrome c Phe-82, i.e. the upfield shift is in the orderpara > meta > ortho. We therefore assign these resonances to the analogous residue, Phe-79. Similarly, the resonances of Rhodopseudomonas viridis ferrocytochrome c2 shown in Table 4 have been assigned to the analogous residue Phe-81. Insufficient signal-to-noise ratio in the spectrum of ferrocytochrome c2 from Rhodospirillum rubrum prevented confirmation by double-resonance experiment of the analogous residue, Phe-93. It is interesting to note that the ortho and meta proton resonances of Phe-79 of Rhodospirillum fulvum iso-1 ferrocytochrome c2 and Phe-8 1 of Rhodopseudomonas viridis ferrocytochrome c2 do not display the same temperature-dependent intensity variation as those of tuna ferrocytochrome c Phe-82. The analogous residue is not present in the remaining bacterial proteins discussed elsewhere in this paper.

Structural Homologq o f Cltochroines c

270 Table 8. 01~c~uri.c~tic.r 01 tynsine, pllenjlulaninr and tryptophan residues in cytochromes c Positions of tyrosin residues

Protein Horse (' Tuna c R. ruhrznn ('1 Rps. viritii,c ( ' 2 R. f u l i w i iso-I

-

~

-

46 46

-

-

31

41

-

('2

48 48 48 47 43

-

52 -.

-

67 61 70 66 62

14 74 -

-

97 97 107 96 93

Positions o f phenylabdnine residues

Positions of tryptophan residues

Assignmcnt of Ti;rosine Resonances The first stage of assignment of resonances to tyrosine residues was carried out as described for horse cytochrome c. As may be seen from Table 1, all the proteins except Pseudomonas arruginosa cytochrome cSj1 contain at least three tyrosine residues. Not all the resonances expected from these residues have been observed in spectra of the reduced proteins at 57 "C. Incomplete first-stage assignment makes confident assignment to specific tyrosine residues more difficult. Sequence comparisons, and the published X-ray structures [ I , 521, suggest that the mitochondrial cytochromes c and the cytochromes c2 from Rhodospirillaceae are very similar. Table 8 shows the presence and absence of tyrosine residues in analogous positions in the sequence of the relevant proteins. Firm assignment of the resonances of Tyr-48 and Tyr-97 of horse cytochrome c have been made. The X-ray structure of tuna cytochrome c indicates that Tyr-67 is very close to, and over the plane of, the heme, and calculations suggest that the resonances arising from Tyr-67 will be shifted upfield out of the aromatic region of the spectrum of the reduced protein [41]. The resonances of two of the three tyrosine residues have been identified in spectra of Rliotlop.seudomonas viridis ferrocytochrome 1 . 2 . and their chemical shift positions are similar to those o f Tyr-48 and Tyr-97 of horse ferrocytochrome c respectively, so have been assigned to the analogous residues, Tyr-47 and Tyr-96. According to the above argument i t is most likely that the unobserved resonances belong to Tyr-66 (analogous to

Tyr-67 of mitochondrial cytochromes c). Spectra of the ferrocytochromes c2 from Rlior~ospirillumruhriim and Rhodospirillumjulvum iso-1 contain tyrosine resonances with similar chemical shifts to those of Tyr-47 of Rhodopseudomonas viridis, and have been assigned to the analogous resonances. Table 5 shows the chemical shift values and assignments of these resonances. It is interesting to note that the temperaturedependent variation in intensity of the resonances of Tyr-48 in spectra of horse ferrocytochrome c is not observed for the analogous resonances in spectra of ferrocytochromes ~ ' from 2 Rhodospirillaceae. The residues appear to be flipping fast above 27 "C in the latter proteins, although other tyrosine resonances do vary in intensity with temperature. The amino-acid sequences of Pscud~onzonasaeruginosa cytochrome c j j 1 [32], Euglena gracilis cytochrome c 5 5 2 [30], Desulfovihrio vulguris cytochrome c 5 5 3 [ 3 3 ] and halotolerant micrococcus cytochrome cs54 are not as closely related to each other or to mitochondrial cytochromes c as are those of cytochrome cz from Rhodospirillaceae, and sequence comparisons do not suggest obviously analogous tyrosine residues. The spectral features of Tyr-48 (and analogous residues) arise from a ring-current shift from the aromatic residue at position 46 (or the equivalent position). None of the other type IC - IE proteins we have studied contain this feature. The only aromatic amino-acid residues in the sequences of Desulfbvibrio vulgaris cytochrome cSs3

27 1

D. J. Cookson, G. R. Moore, R. C. Pill, R. J. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, and J. Le Gall

Tabelle 9. ( A ) Relative sequence position of tryptophan and hem-binding residues; ( B ) occurrence of tryptophan and methioninr residues In (B), residues in bold-face type are those thought to provide the 6th iron ligand Protein

(A) Position of residues cys-

Horse c Tuna c R. rubrum c2 Rps. viridis c~ P. aeruginosa cs5 E. gracilis c 5 5 2

XXX-XXX-CYS-11,s

14 14 14 13 12 10

Met

TrP

Met 80 80 91 79 61

56

59 59 62 58 56 59

M65 M65

-

w59 w59 W62 W58

M52

-

-

M56

W56 w59

M58 M61

M80 M8O M91 M79 M75

M47

-

-

-

18 18 18 17 16 14

(B) Positions of tryptophan (W) and methionine (M) Horse c Tuna c R. rubrum c2 Rps. viridis c2 R. fulvum iso-I c2 P.aeruginosa c551 E. gracilis c552 D . vulgaris c553 Halotolerant micrococcus cS54 a

-

-

M55

-

-

-

-

M64 M60,63”

M79 -

The methionine ligand in halotolerant micrococcus c554 is either Met-60 or Met-63

and halotolerant micrococcus cytochrome c554 are tyrosine residues (Table 1). The resonances of these residues have been assigned in some oxidation states for both these proteins. Some of these resonances have a temperature-dependent intensity variation. Their chemical shift positions and coupling patterns will be published shortly. Assignment OJ the Resonances of Aliphatic Groups Close to the Heme meso Protons

A number of one-proton multiplets have been observed in spectra of all these reduced proteins, between 4.8 ppm and 6.4 ppm (Fig. 1,2). In well-resolved spectra these often appear as quartets, and many are coupled to three-proton doublets occurring between 1.9 ppm and 2.6 ppm. This coupling pattern can only arise from a CH-CI& group, that is from alanine or threonine residues, or from the thioether heme substituents. The latter possibility was discounted by us in the case of horse ferrocytochrome c because the thioether methyl resonances have been positively identified at 0.3 ppm and 1.4 ppm [35].However these assignments are not generally accepted. Many of the one-proton resonances show a negative nuclear Overhauser enhancement to individual heme meso protons. The relevant data are shown in Table 6. Additionally these one-proton and threeproton resonances suffer a large downfield shift from their primary positions. These data place the groups close to, and in the plane of, the heme. Assignment to specific amino-acid residues cannot yet be made

+

+

for these resonances in spectra of bacterial proteins, but they must arise from alanine, threonine or thioether residues close to the heme.

DISCUSSION In this section we first compare the properties of the resonance of mitochondria1 and bacterial type I cytochromes c that have been assigned in the previous section, then proceed to discuss critically the conclusions thay may be drawn and hypotheses postulated concerning the solution structures and other properties of these proteins.

COMMON RESONANCES

Heme Axial Ligunds

The resonances listed in Table 2 confirm that all these proteins have histidine and methionine residues as the 5th and 6th axial ligands to the iron. This fact defines one basic common feature of the folding of these proteins : that the polypeptide chain is bound to the heme at four points, three close together near the N-terminus (the heme-binding site), and a fourth point at the position of the methionine residue. However, the sequence positions indicated in Table 9 show how much the location or this methionine residue may vary along the sequence, with large changes in the length of the loop between the histidine and methionine residues.

212

Structural Homology of Cytochroines c

Trjpptoplzan-j!J2

A tryptophan residue occurs at the same position in the sequences of all mitochondria1 cytochroines so far studied [53]. Its resonance positions in spectra of these proteins depend on the oxidation state of the iron, suggesting that Prp-59 is near to the heme, a result in accord with the X-ray structure of tuna cytochrome (' [8.9]. The resonances of Trp-56 of Pseudonio~iu.~ ~ ~ ~ r i ~ g i t icytochrome o.ru cssl possess a similar dependence on oxidation state [17]. Table 3 shows the positions of these tryptophan resonances and those of analogous resonances from other diamagnetic cytochromes c. It is immediately seen that these resonances are considerably shifted from their primary positions and that the shifts are very similar for each protein. This argues strongly for a similar tryptophan environment in each protein. We therefore conclude that not only is this region of the protein unaffected by change in oxidation state (compare ferrocytochrome c and cobalticytochrome c in Table 3), but that it is very similar in many different proteins. The heme group possesses 8-fold spatial degeneracy with respect to its ring-current field (but is of course asymmetric with respect to the protein). Therefore the shifts observed on the tryptophan resonances, although indicating a similar tryptophan.heme orientation, d o not define a unique protein fold. Table 9 shows the relative positions in the sequence of these tryptophan residues. The posilion of the inethionine ligand residue in the sequences of cytochromes c varies considerably with respect to the heme-binding site (Cys-Xxx-Xxx-CysHis) and the tryptophan residue. In these proteins the tryptophan is separated from the heme-binding site by 40 -- 45 residues, while the methionine ligand occurs as many as 30 residues to the C-terminus side of the tryptophan to as little as six in the case of Pscwdonio7iu.s a w i g i r m a cytochrome cs51, and actually precedes the tryptophan in the sequence of Euglena gracilis cytochrome 1 ' j 5 2 . In view of this it is difficult to see how the fold can be the same, especially in the case of Eu,rrlmii graci1i.s cytochrome c 5 5 2 . However, the tryptophawheine orientation must be very similar in all these proteins. This evidence suggests that the analogous tryptophans play an important functional role. The available X-ray analyses for type I cytochromes c show that this tryptophan residue is involved in hydrogen bonding to the heme propionate substituents [7-~9]. It is interesting to note that A'-formyl modification of Trp-59 in horse cytochrome c ruptures the ironsulphur bond. and radically alters the properties of the protein [54,55], suggesting that this hydrogenbonding network is important for the protein's stability. (2

' \equenw

niinihci iel'eri t o horse cytochrome c

Two cytochromes c from Rhodospirillaceae which do not contain a tryptophan residue have been isolated, but these tryptophan-free proteins have only been found in systems which also have a tryptophan-containing iso-protein. We return later to cytochromes ~ 5 5 . 3 and ('554 which are the only remaining tryptophan-free type I cytochromes c so far discovered.

PIze~ylalanine-822 The data shown in Table 4 shou, that this conserved phenylalanine residue in mitochondria1 cytochromes c and cytochromes c2 from Rhodospirillaceae occupies a similar position in space over the heme in each protein. The upfield shift experienced by the resonances, in the order para > nictu > ortlio shows that the orientation is similar. This evidence is in agreement with the most recently published data on the structure of tuna cytochrome c and Rliocko.s~~irillurn ruhruni cytochroine c2 [8- lo]. While it has been shown that the flipping of Phe-82 in horse cytochrome c about its C-P- C-;; axis is restricted, the corresponding residues in R/iodo.s/~iri/lum,fuhuni iso-1 and Rhodopscudonzoiius viridis cytochromes c2 give rise to the type of resonance pattern associated with fast flipping. It is possible that the smaller chemical shift difference between each oi.fiio and meta proton could account for this, or that the rotation of these phenylalanine residues is less restricted. The former explanation is consistent with the observed ring-current shifts experienced by the phenylalanine resonances shown in Table 4. For the para proton resonances the shift from the primary position is in the order:

R.fUlvun? < horse = iso-1 2 Rps. ortho and ineta observed :

viridis at all temperatures

tuna < R. rlrhrLlt?1

only at high not temperaobserved. tures

The fact that the para proton resonance is observed at all temperatures indicates that no anisotropic motion of the phenylalanine ring occurs (or that any motion is rapid). The absence of a corresponding phenylalanine residue from the remainder of the bacterial proteins rules out an essential structural or functional role for this residue. If an aromatic or merell. bulky residue is necessary in this position, perhaps these latter proteins employ an alternative residue. A two-proton doublet in the spectra of Pseudonionas aerugiriosa cytochrome c551, arising from a tyrosine or phenylalanine residue but which has not yet been assigned, may well come from such a residue. I t experiences a

D. J. Cookson, G. R. Moore, R. C. Pitt, R. J. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, andJ. Le Gall

large upfield shift in the reduced protein, and is shifted downfield on oxidation [17]. These are the characteristics of a resonance arising from a residue in a similar position over the plane of the heme. It is interesting to note that this protein only contains tyrosine or phenylalanine groups at positions 7, 34 and 27 and thus the residue giving rise to these signals is not homologous in the primary structure to Phe-822. Tyrosine-4S2 Despite the fact that each mitochondrial cytochrome c and bacterial cytochrome c2 possesses either a phenylalanine or tyrosine residue at a position analogous to that of Phe-46 of horse cytochrome c, and a tyrosine residue at a position analogous to that of Tyr-48 of horse cytochrome c, only the resonances of horse and tuna cytochrome c listed in Table 5 have a temperature dependence associated with restricted rotation over the range 27- 77 “C [21,43]. From this fact it is reasonable to deduce that the restrictions on the mobility of this residue (Tyr-4g2) are not so great in the bacterial proteins, indicating that the packing of neighbouring groups in this region of the protein is looser. Aliphatic Groups Near the Heme

The data shown in Table 6 indicate that in each of these proteins two aliphatic residues are packed sufficiently closely to the heme that their methylene protons are in Van der Waals contact with two heme meso protons. That this should be so each of these proteins, which have considerable differences in primary structure, suggests that this feature is general to cytochrome c of type I. The groups could be from an amino acid or a thioether.

GENERAL DISCUSSION

Static Structures

Representatives of c-class cytochromes from vertebrates, Rhodospirillaceae and Pseudomonads have been investigated by X-ray crystallographic methods, and their ‘static’ structures determined. It has been suggested that type I cytochromes c have a common structural feature, termed the ‘cytochrome fold’ [12]. The NMR studies reported here show that the conformation of mitochondrial cytochromes c and cytochromes c2 from Rhodospirillaceae in solution are very similar, and consistent with the published X-ray data. The spatial distribution of a large enough fraction of the amino-acid content (e.g. 12 out of 107 residues in the case of Rhodopseudomonas viridis cytochrome c2) has been determined for this conclusion

273

to be stated with confidence. The striking similarity, in terms of both sequence and structure, supports the recent suggestion that mitochondrial cytochromes c should be classified as cytochromes c2 [I, 1 11. For Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome ~ 5 5 2it is found that many features of the heme environment are similar to those of the above proteins, but the massive deletions of more than 20 amino-acid residues between the heme binding site and the methionine ligand must induce a change in the folding of the polypeptide chain, which must be fundamental in the case of Euglenagracilis cytochrome c552, in which the methionine and tryptophan residues occur in the reverse of the normal sequence. For halotolerant micrococcus cytochrome c554 and Desulfovihrio vulgaris cytochrome c553 there is only limited evidence from this study for structural homology. The heme-binding site and methionine ligand are the same, and aliphatic groups are in Van der Waals contact with heme meso protons, but the only aromatic amino acids in their sequences are tyrosine residues. At least some of these tyrosines are far from the heme. This, and the lower total incidence of aromatic residues, suggests that the heme environment is less hydrophobic. It is interesting to note that these proteins have much lower redox potentials than other type 1 cytochromes c. The results of these studies tend to support the conclusion that rather than the protein fold, the environment of the iron atom is the most strongly conserved feature of these proteins, and that when the two are incompatible, as in the case of Euglena gracilis cytochrome c552, it is the heme environment which is preserved. Dynamic Structures

Where dynamic information has been obtained it sheds new light on the protein structures. Thus we observe that while mitochondrial cytochromes c and bacterial cytochromes c2 have very similar ‘static’ structures, they have different ‘dynamic’ structures. This is clearly shown in the case of the T ~ r - 4 8 ~ residue. In mitochondrial cytochromes c the motion of this residue is severely hindered by the surrounding groups, but we do not observe any restriction on flipping in bacterial cytochromes c2. However. some resonances whose lineshape and intensity are temperature-dependent are observed in spectra of the latter proteins, indicating that the motion of some residues is restricted. No restricted motion has been detected for Pseudomonas aeruginosa cytochrome c 5 5 1 in solution. It is apparent that several of the tyrosine residues in Desulfovibrio vulgaris cytochrome c553 and halotolerant micrococcus cytochrome c554 have restricted motion.

274

Restrictions on motion reflect the rigidity of a protein's structure, which we observe to vary considerably within this class of protein. Redo Y Potmtuls In the course of these studies, it was noticed that a relationship existed between the redox potential of a type I cytochrome c and the chemical shift position of its methionine ligand resonances in spectra of the reduced form This correlation has been interpreted as follows [56]. Increases in the upfield ring-current shift of the methionine ligand methyl resonances in spectra of reduced cytochromes c are due to a decrease in the iron-sulphur bond lengths, which arises from an increase in the ability of the sulphur to donate electrons to the iron. This will lead to a stabilization of the iron(II1) state and hence a decrease in redox potential. The ability of the methionine ligdnd to donate electrons I S modified by the protein structure. Structure mcI Sryiwtiw Homology

We suggest that. contrary to what one might expect, it appears that the most strongly conserved features of these proteins are the heme binding site (Cys-Xxx-Xxx-Cys-His) and certain of the aromatic amino acid residues, while the sequence position of the methionine ligand is more labile. Table 9 (B) shows the sequence positions of the methionine residues in these proteins. and the conserved tryptophan residue. The histidine-tryptophan separation is constant whilst the histidine-methionine separation is variable.

CONCL LJ S ION S 1. These studies demonstrate that N M R is a sensitive and powerful tool for comparative structural investigations ol' proteins. 2. We conlude from the data presented here that the heme environment of the high-redox-potential cytochromes c is very similar, and hydrophobic in na t Lire. 3. The lower-potential proteins we have investigated show fewer similarities. 4. A direct relationship between redox potential and the environment of the methionine ligand has been demonstrated for all of the above proteins. Wc thank thc Science Research Council, the Royal Society and the Mcdical Research Council for financial support. R. J.P. W. is ;I member of the Oxford Enzyme Group.

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D. J. Cookson, G . R. Moore, R. C. Pitt, and R. J. P. Williams*, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, Great Britain, OX1 3QR I. D. Campbell, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, Great Britain, OX1 3QU R. B. Ambler, Department of Molecular Biology, University of Edinburgh, Kings Building, Mayfield Road, Edinburgh, Great Britain, EH9 3JR M. Bruschi and J. Le Gall, Laboratoire de Chimie Bacterienne du C.N.R.S., 31 Chemin Joseph-Aiguier, F-13274 Marseille-Cedex-2, France ~~

*

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To whom correspondence should be addressed.

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