The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high-resolution 1H-NMR spectroscopy

Eur. J . Biochem. 192,715-721 (1990) 0 FEBS 1990

The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high-resolution 'H-NMR spectroscopy David WHITFORD', David W. CONCAR', Nigel C. VEITCH' and Robert J. P. WILLIAMS'

Department of Biochemistry and Inorganic Chemistry Laboratory, University of Oxford, England

(Received February 19/June 11, 1990) - EJB YO 0171

The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one- and two-dimensional 'H-NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/bs molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome h5 with increasing ratios of ferricytochrome c indicate the formation of binary 1 : 1 complexes and ternary 1 : 2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome h5 are consistent with stoichiometries of 1 : 1 and 1 : 2 for complexes formed between cytochromes h5 and c. Surprisingly, in the 1 : 1 complex, mobility is shown to be a function of ionic strength. Two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the a-CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.

Cytochrome b5 is found in the microsomes of eukaryotic cells as a membrane-bound component of the fatty acid de- saturase system. The protein has a hydrophilic domain containing approximately 90 amino acids as well as a smaller hydrophobic portion of 30 -40 amino acids which provides a C-terminal anchor to the membrane. In most biochemical studies of cytochrome bs, workers have used the hydrophilic fragment of approximately 86 amino acids released by limited proteolytic digestion of bovine liver microsomes [l -31. Do- mains with high sequence similarity to the bovine microsomal protein have been identified in a wide range of enzymes underscoring the importance of this structure as an electron transfer motif [4-71.

The parameters influencing interactions between electron transfer proteins can only be understood by obtaining infor- mation on their respective molecular structures. Fortunately high-resolution structures are available for cytochrome b5 and a number of variants of cytochrome c [8-111. X-ray crystallography of cytochrome hs , at a resolution of 0.2 nm, has revealed the oxidised protein to be a cylindrical molecule containing six CI helices and five strands [12]. The haem centre, located in a hydrophobic pocket and coordinated to two histidine ligands, is surrounded by a cluster of 11, out of a total of 18, negatively charged groups. The consensus is that these charged groups, distributed asymmetrically about the surface of cytochrome bs, enable binding to both small molecules and proteins including cytochrome c [13, 141. The important role of these charged groups is supported by the

Correspondence to D. Whitford, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, England

Abbreviations. NOESY, nuclear Overhauser effect spectroscopy; DQF-COSY, double quantum filter correlated spectroscopy.

observation of rates of electron transfer that are greater with positively charged reactants and show a marked dependency on ionic strength [15, 161. Computer modelling studies of the complex formed between cytochrome b5 and cytochrome c, which like cytochrome b5 is a highly charged protein, suggested specific salt bridges between lysines 13, 27, 72 and 79 of cytochrome c and negatively charged groups on cytochrome b5 (Glu44, Glu48, Asp60 and the exposed haem propionate) promote protein association [17, 181. Experimen- tal support for the proposed structure of the binary complex has come from optical spectroscopy showing the association to be dependent on both ionic strength and pH, and from chemical modification studies of both reaction partners that further emphasised the involvement of charged surface residues in complex formation [I9 - 231. Although microsomal cytochrome b5 and cytochrome c appear not to be physiologi- cal redox partners, stopped-flow and pulsed radiolysis measurements indicate rapid electron transfer between the two proteins and this couple represents a useful system in which to study complex formation and first-order electron transfer

Despite much indirect evidence for the proposed interaction between cytochrome c and microsomal cytochrome bs, direct demonstration, for example by crystallisation of the cytochrome b5/c complex, has not as yet been achieved. In the absence of such a structure, NMR represents one of the few direct methods available to investigate the structure and dynamics of protein -protein interactions. Cytochrome c has been the subject of many NMR studies and from a combi- nation of one- and two-dimensional methods sequence- specific assignments are available for both wild-type and site- directed protein variants [25 - 281. For cytochrome h5 hyperfine-shifted haem and haem ligand resonances, together

~241.

716

with some of the aromatic side chains, have been identified [29 - 341; more recent studies of cytochrome b5 have extended the number of assignments with over 60% of all protons identified for the oxidised and reduced forms of the protein [35, 361.

The presence of extensive NMR data for both redox partners has allowed the cytochrome h5/c complex to be studied. Determination of the stoichiometry of the complex using NMR led to the suggestion that a ternary complex was formed and contradicted the general validity of the Salemme electrostatic model [37]. However, other NMR studies have failed to detect ternary complex formation and supported a binary complex as implied by both optical spectroscopy and analytical ultracentrifugation [19, 21, 381. It is clear that the present knowledge of the stoichiometry of the cytochrome h5/c complex is insufficient to analyse critically the validity of the Salemme model.

Recently, dynamics of protein - protein interactions have been proposed as important in facilitating complex formation. This has led to the use of computer models to evaluate the role of structural fluctuations in the association of cytochromes c and b5 and intra-complex electron transfer [39]. The structure, stoichiometry and mobility of protein complexes is central to an understanding of long-range biological electron transfer and the role of cytochrome b5 in vivo. In this paper one- and two-dimensional NMR techniques are used to examine both the stoichiometry and dynamics of the complex formed between ferricytochrome b5 and ferricytochrome c and to assess the accuracy of current models of interaction between these proteins.

METHODS

Protein isolation

Cytochrome c was obtained from Sigma Chemical Co (type VI) and purified by cation-exchange chromatography on CM-32 cellulose to remove deaminated protein as described previously [40]. The purified protein was concentrated and exchanged into phosphate buffer and stored at - 20 "C.

Cytochrome b5 was extracted as a hydrophilic fragment obtained by proteolytic digestion of bovine liver microsomes using the outline of methods described previously [16, 41,421. Fresh bovine liver, washed with a small volume of 0.25 M sucrose, 1 mM EDTA pH 7.4 to remove excess haemoglobin, was homogenised (500-g portions/ M 1.5 1 sucrose solution) and centrifuged at 16000 x g for 10 min to remove heavy cellular debris. The light-brown/red supernatant was decanted and ammonium sulphate added to 40% saturation. The pH was kept at 7.2 and the solution left at 4°C for at least 20 min. Centrifugation at 20000 x gfor 40 min yielded the microsomes as a light-brown pellet which was resuspended in a minimal volume of 0.1 M Tris/HCl pH 8.1, 1 mM EDTA and dialysed extensively against distilled water to remove ammonium sulphate. The microsomes were frozen and stored at - 70 "C.

To 1 1 microsomes, 500 mg trypsin (Sigma type I) in 15 ml 1 mM HC1 and 70 mg tosylphenylalanylchloromethane were added and the mixture left stirring for M 48 h at pH 7.6 and 4 'C. Membranous material was precipitated by the addition of 40% ammonium sulphate and removed by centrifugation 16000 x g for 30 min. The red-orange supernatant was re- tained and synthetic trypsin inhibitor (p-nitrophenyl-p- guanidinobenzoate hydrochloride) added to the solution at a molar concentration three times that of trypsin. The mixture was left for 20 min and then dialysed for approximately 12 -

18 h to remove ammonium sulphate. Precipitated material was removed by centrifugation before loading the orange-red solution onto a DE-52 anion-exchange column (2.5 x 60 cm) equilibrated in 20 mM phosphate pH 7.0. The column was washed with 1 120 mM phosphate buffer and eluted at a flow rate of 40 ml h- ' with a linear gradient of KCI formed by mixing 500ml each of 20mM buffer and 0.4M KCl. Cytochrome fractions were pooled and concentrated by ultrafiltration (Amicon 8400, YM5 filter) to a volume of about 3-5ml before applying to a Sephadex G-75 column (2.5 x 60 cm) equilibrated in 20 mM phosphate pH 7.0. The cytochrome was eluted in 20 mM phosphate buffer and all fractions with an A413/A280 ratio greater than 5.5 were pooled and used in NMR experiments.

Cytochrome b5 was reclaimed following NMR experiments by diluting mixtures of the protein contaminated with cytochrome c and applying to short (1 x 5 cm) DE-52 columns. Recovery of cytochrome b5 was estimated to be greater than 95%. Protein concentrations were estimated from the absorbance at 412.5 nm in the oxidised state and assuming an absorption coefficient of 117 mM-' cm-' [3].

NMR spectrometry

NMR spectra were recorded on a Bruker AM-600 spectrometer equipped with an Aspect 3000 computer. All spectra were recorded at 300 K, involved the collection of 512-2048 transients, a sweep width of 50 kHz and storage in 16 K mem- ory. Free induction decays were Fourier transformed without any form of resolution enhancement to preserve natural linewidths. Two-dimensional NMR experiments were recorded on a home-built 500-MHz spectrometer equipped with a GE/Nicolet 1280 data acquisition system. Spectra were col- lected over a sweep width of 12.5 kHz with the offset positioned in the centre of the spectrum and the residual water resonance suppressed by presaturation. Phase-sensitive double-quantum-filtered correlation spectroscopy (DQF- COSY) [43] and nuclear Overhauser effect spectroscopy (NOESY) [44] were carried out using the methods of States et al. [45] and with standard phase-cycling procedures. The data acquired was resolution-enhanced in t2 using double-ex- ponential multiplication and trapezoidal functions and in tl using the former together with a shifted sine bell function. A mixing time of 130 ms was used in the NOESY experiments. All chemical shifts are quoted relative to 2,2-dimethyl- silapentane-5-sulphonic acid.

Samples were prepared for NMR experiments by ultrafiltration using an Amicon microconcentrator (molecular mass cut off 10 kDa) in 10 mM phosphate/NaOD pH* 7.2. Stock solutions of 1 M phosphate pH 7.2 were added directly to the NMR cell to elevate the ionic strength of the solution. In view of the known ionic strength and pH dependency of complex formation, each sample was made up in 10 mM phosphate pH 7.2 and to contain 0.5 mM ferricytochrome b5. Samples therefore differed only in the amount of ferricytochrome c present.

RESULTS

Fig. 1 shows the hyperfine-shifted regions of isolated ferricytochrome c and ferricytochrome b5 between 10 - 40 ppm. The large secondary shifts of these resonances, which have been assigned chiefly to haein and haem ligand groups, result from large contact and pseudocontact interactions with the

71 7

30.00 25.00 20 .00 15.00 chemical shift (PPW

Fig. 1. The hyperfine-shifted resonances of ferricytochrome c, ferri- cytochrorne b5 unda mixture of the twoproteins. Spectra were recorded at 600 MHz, 300 K in 20 mM phosphate pH 7.2. Resonances at 30.4, 27.3, 17.9 and 16.2 ppm have been attributed to protons of the por- phyrin ring of a minor isomeric form of cytochrome b5 representing less than 10% of the total protein [29, 301. It is worth noting that these resonanccs are observed within the complex with cytochrome c with no obvious change in their intensity or ratio to the major isomer. Complex formation is thus unaltered by the presence of major and minor isomers of cytochrome b5

oxidised, paramagnetic, haem iron centres of both proteins. The addition of ferricytochrome c to ferricytochrome b5 results in a spectrum that represents the composite of the spectra of the two individual proteins. The hyperfine-shifted resonances of cytochrome b5 show small but significant changes in both chemical shifts ( A 6 = 0.05-0.3) and linewidths. The line-broadening is not confined to the hyperfine-shifted resonances but is observed throughout the spectrum and is expected in view of the known formation of protein complexes between the redox partners resulting from the increased rotational correlation time for the cytochrome b5/c complex.

Close inspection of the whole spectrum of the ferricytochrome b,/c mixtures reveals that most resonances of the aliphatic and aromatic regions show no significant changes in their chemical shifts. However, a number of hyperfine-shifted resonances do show small chemical shift perturbations in- dicating that the magnetic micro-environment of certain proton groups are affected by complex formation. Measurements of the changes in the chemical shifts of eight hyperfine-shifted resonances of ferricytochrome b5 at molar ratios of cytochrome c ranging over 0.1 -4.0 are shown in Fig. 2. The titration profiles show two distinct phases: a linear dependency on the cytochrome clcytochrome b5 ratio up to the addition of one molar equivalent followed by more gradual shift changes for resonances assigned to haem C2 methyl (formerly designated as haem methyl 1, 11.6 ppm), haem C17 propionate CI ( C ~ ~ C I , 18.9 ppm), haem C7 methyl minor (C7m, 30.4 ppm), C3 vinyl-2a (C3 V2a, 27.0 ppm) and the haem C12 methyl major and minor isomers (C12, 21.7 ppm and 26.8 ppm). It was also apparent that some resonances were significantly altered only by the addition of the first molar equivalent of ferricytochrome c, e. g. haem C7 methyl (C7,

21.5

21.4 d

0 1 2 3 4 [FemCc] I [Femb,]

[FemCc]lIFem bsl

Fig. 2. The titration of the chemical shifts of resonances of ,ferricytochrome b5 with increasing ferricytochrome c. All titrations were carried out in 20 mM potassium phosphate pH 7.2, 300 K and a1 spectrometer frequency of 600 MHz. The resonances labelled a- i are assigned to the following groups [30,32]: a = haem C7 methyl minor isomer, b = haem C18 methyl minor isomer, c = haem C2 vinyl-2- a, d = haem C12 methyl, e = haem C17 propionatc-a-CH, f = haem C7 methyl, g = haem C2 methyl, h = haem C2-F-vinyl cis, i = haem C2-F-vinyl trans. [Fe"'Cc] = ferricytochrome c, [Fe"'b,] = ferricytochrome h5

14.4 ppm) and the C2 vinyl-2P protons (C2 V2p, -6.1, -6.9 ppm). It is interesting to note that the chemical shift perturbations assigned to the CI proton of haem C17 propionate are strongly influenced by ferricytochrome c. This is perhaps not unexpected in view of the observation that one or more of the haem propionates is known from esterification studies to influence the binding of cytochrome c to cytochrome b5 [15, 201. A qualitatively similar chemical shift profile with increasing cytochrome c concentration was obtained when the titration was performed in a background bufferlelectrolyte of 10 mM cacodylate/NaOH pH 7.2 (results not included). One further test of the dependence of complex formation on the solution conditions involved increasing the concentration of cytochrome b5 to twice that of the titration shown in Fig. 2 (cytochrome b5 = 1 .O mM). The variations in chemical shifts were very similar to those performed at lower protein concentrations but reached saturation close to about two molar equivalents. This result is consistent with increased binding of cytochrome c to cytochrome b5 at an additional second site at these protein concentrations.

Fig. 1 shows considerable peak overlap occurs in mixtures of ferricytochrome c and b5 and that at high cytochrome clb, ratios (clh, > 5 ) very few resolved cytochrome b5 resonances exist. Consequently, any analysis of protein association, from the concomitant increases in resonance linewidth, is restricted to a few selected resonances. The resonance at 21.7 ppm assigned to haem C12 methyl (formerly designated haem methyl 5) of ferricytochrome b5 represents one well-resolved peak for which the linewidth can be measured in the presence of a 10-fold molar excess of cytochrome c. The increase in the linewidth of the haem C12 methyl with increasing cytochrome c/b5 ratios (Fig. 3) shows two major regions. An initial large increase in linewidth up to one molar equivalent of cytochrome c is followed by further line broadening which increases more slowly with higher cytochrome c/b5 ratios. An eventual saturation of the linewidth under the present solution conditions was observed at a 10-fold molar excess of cytochrome c. The cis and trans vinyl-2-CH groups at -6.8 and - 7.0 ppm also remain resolved throughout the titration and show similar linewidth increases above one molar

71 8

P 2 70

3 f 6 0 ~

80

. ooo

Cyt b,

Haem Methyl 5 , O

40 "L 0 1 2 3 4 5 6 7 8 9 10

[Cytcl/[C~tbsl

Fig. 3. The effect of increasing ferricytochrome c concentration on the linewidth of haem C12 methyl ojferricytochrome b5. Conditions were as for Fig. 2. [Cyt c]/[Cyt b5] = molar ratio of ferricytochrome c to ferricytochrome b5

40 20 40 60 80 100 120 140

[phosphate] (mM )

Fig. 4. The linewidth of haem C12 methyl resonance of ferricytochrome b5 measured in the presence of an equimolar ferricytochrome c concentration as a function of ionic strength. The 1 : 1 mixture contained 0.5 mM ferricytochrome c and 0.5 mM ferricytochrome b5 in potassium phosphate pH 7.1. All measurements were performed at 600 MHz and 300 K. The linewidths throughout a given titration were measurable to z 1.5 Hz. The linewidths from one titration to another are very sensitive to both temperature and solution pH lead- ing to a variation of == 2 Hz in the natural linewidth of the haem C12 methyl of ferricytochrome b5

equivalent of cytochrome c. There is therefore evidence, from both the line broadening of these resonances and their chemical shifts, of additional protein association above cytochrome clb, ratios of 1.

The binding of a second molecule of cytochrome c to the surface of cytochrome b5 would appear to indicate the presence of trimeric complexes in addition to binary 1 : 1 aggre- gates. To determine the possible states of aggregation of the cytochrome b,/c complexes the linewidth of the well-resolved haem C12 methyl resonance of ferricytochrome b5 (21.7 ppm) was measured in an equimolar (1 : 1) mixture of cytochromes b5 and c as a function of ionic strength (Fig. 4). The linewidth at ionic strengths less than 20 mM was approximately 70 Hz but diminished rapidly, upon elevating the background electrolyte level, reaching a plateau (linewidth M 54 Hz) that was maintained between 25 - 50 mM phosphate before decreasing in two further sequential steps to a linewidth of 45 Hz above

120 mM phosphate. This latter linewidth is identical to the linewidth of haem C12 methyl in free cytochrome b5 measured at similar solution conditions. At ionic strengths between 70 - 100 mM a linewidth of 48 Hz was observed that remained constant with increasing ionic strength and was consistently greater than that observed for the uncomplexed, free, protein. For each plateau the intrinsic linewidth could be measured reproducibly to within 1.5 Hz. The titration profile therefore shows a narrowing of the linewidth within the dimeric 1 : 1 complex that may be equated with increased intra-complex mobility.

The recent modelling of the dynamics of protein interaction between cytochrome c and b5 invoked a possible role for the evolutionarily invariant Phe82 of cytochrome c at the protein interface [39]. In view of the large number of assignments made for resonances of both cytochrome c and b5, it was possible to estimate the effect of protein binding on specific amino acid resonances by two-dimensional NMR methods (DQF-COSY and NOESY). A protein sample containing 4 m M protein was made up in 10 mM phosphate pH 7.2. Under these conditions it was assumed that the proteins were completely associated and that possible binding of cytochrome c at a secondary site was negligible. The aromatic region of the DQF-COSY spectrum for an equimolar mixture of the two proteins showed no major chemical shift changes (Fig. 5). Significantly, the resonance at 6.10 ppm previously assigned to the Phe82 ortholmeta protons of native ferricytochrome c was found to be unperturbed by complex formation as was the cross peak of Thr78, another residue located near to the proposed binding domain. In the upfield methyl region, DQF-COSY of the complex showed that none of the cross peaks are significantly shifted from their positions in free ferricytochrome c and ferricytochrome h5. The only cross peaks substantially altered by complex formation were those attributable to the haem propionates of cytochrome b5 as indicated in Table 1. These observations were supported by NOESY spectra (Fig. 5B) which show that in the presence of equimolar cytochrome b5 the aromatic ring of Phe82 by cytochrome c maintains the NOE connectivities of Ile85, Leu68 and thio-ether 2, present in isolated cytochrome c. Interestingly the relative intensities of these NOESY cross peaks are enhanced by complex formation suggesting that the increase in rotational correlation time which accompanies complex formation leads to more effective interproton cross- relaxation. The two-dimensional spectra are therefore in agreement with the data from chemical shift titrations, suggesting that major conformational changes in the structure of cytochrome b5 are absent upon complex formation with cytochrome c.

DISCUSSION

The chemical shift titration data of hyperfine-shifted resonances of ferricytochrome h5 are consistent with a single high-affinity cytochrome c binding site on the surface of cytochrome b5 and at least one secondary binding site of lower affinity. The titration profiles of Figs 2 and 3 suggest that under appropriate experimental conditions the high-affinity site becomes fully occupied before appreciable binding occurs at the secondary site. Thus at low protein concentrations the binary 1 : 1 complex dominates with the ternary complex being formed only at higher protein concentrations. From the titration profile shown for the resonance assigned to haem C17 propionate a (18.9 ppm) and from a knowledge of the ultimate

719

6.5 6.0 5.5 Chemical Shift w m )

:m:s

185 185 B ffill HD

F82 HZ

185 185 TE L68

5.8

6.0

6.2

F82 HZ

F82 HD &HE

TE L68

5.8

6.0

6.2

F82 HD &HE

2.0 1 .o 0.0 -1.0 -2.0

Chemical Shift (Ppm)

Fig. 5 . Two-dimensional ' H - N M R spectra of the binary complex of ferricytochrome b5 andferricytochrome c. (A) The aromatic region of a phase-sensitive DQF-COSY spectrum recorded at 500 MHz for an equimolar mixture of cytochrome h5 and cytochrome c. The sample contained 2 mM ferricytochrome b5 plus 2 mM ferricytochrome c in 10 mM potassium phosphate pH 7.2 at 303 K. (B) Phase-sensitive NOESY spectrum of an equimolar mixture of cytochrome b5 and cytochrome c. The pattern of NOE connectivities from the aromatic protons of ferricytochrome c Phe82 is illustrated. TE2 denotes the haem thio-ether 2 methyl protons. Note that a similar set of NOE connectivities is seen to that of isolated ferricytochrome c

Table 1 . The chemical shift of selected resonances of ferricytochrome b5 in the presence and absence of equimolar,ferricytochrome c

Resonance Chemical shift in cytochrome b5

free bound

PPm

6 P-CH 6 P-CH 7 /I-CH

-1.53 -1.41 -0.75 -0.69

1.66 1.73

stoichiometry of the protein complex, an association constant can be estimated for the low-affinity site by measuring the response of the chemical shift of this resonance to cytochrome c added in excess of one molar equivalent. This approach yielded an association constant of 8.9 (0.3) x lo3 M-' i n- dicating that binding at the secondary site is at least an order of magnitude less than that estimated for the primary site (K, z lo5 M-l , 10 mM phosphate pH 7.0,297 K). The pres-

ence of ternary complexes is in contrast to the simple 1 : l model proposed on the basis of surface charge comple- mentarity for interactions of proteins with cytochrome b5 [13, 14,461. It is probable that the many negatively charged groups of cytochrome b5 present numerous potential binding domains for both proteins and inorganic reagents. Earlier data obtained from chemical shift titrations were consistent with the formation of ternary complexes between two cytochrome c molecules and cytochrome b5 [37]. However this study was based on the chemical shift dependency of three resonances at a single cytochrome b5 concentration and did not distinguish between primary and secondary binding. Additionally ternary complexes of cytochrome b5 with small molecules and cytochrome c have been detected previously in solution [47, 481.

The NMR results obtained here and in previous studies indicate the formation of ternary complexes between cytochromes b5 and c and are therefore in contrast to results obtained by optical spectroscopy [19]. The NMR experiments show primary and secondary binding at a range of protein and electrolyte concentrations (0.5 -2.0 mM protein, 298 K, pH 7.2, phosphate, Z z 10 mM) and it is hard to compare with measurements of the change in absorbance at 416 nm upon mixing each protein (1 - 10 pM protein, 298 K, pH 7.0, phosphate Z = 1 mM).The results may be reconciled if it is assumed that the difference absorbance maxima are relatively insensi- tive to binding at the low-affinity site. In view of the small changes in absorbance due to primary binding (dA < 0.04), it is unlikely that secondary binding would provide discernable optical effects. Additionally hydrodynamic techniques such as analytical centrifugation and gel filtration chromatography are unlikely to detect ternary complexes under the usual experimental conditions of high ionic strength and low protein concentrations where the proportion of secondary binding sites occupied is low.

The changes in the chemical shift and linewidth of the C12 haem methyl (21.7 ppm) that occur in the presence of cytochrome c can most readily be accounted for by involving two binding sites on the surface of cytochrome b5 for cytochrome c. However the action of increasing the ionic strength suggests that the linewidth or rotational mobility of ferricytochrome b5 may be determined by both its aggregation state and by externally induced changes in the dynamic status of the complex. At low ionic strength the decrease in mobility of cytochrome b5 in the presence of equimolar cytochrome c suggests that the binary complex has a tendency to self associ- ate which diminishes as the concentration of background electrolyte increases. Eventually a binary complex is formed which remains stable between 25 - 50 mM phosphate and gave a linewidth for the haem C12 methyl of 55 Hz. This value is close to that expected for the formation of a binary complex which should, to a first approximation, double the diamag- netic contribution to the linewidth. The relationship between molecular mass and linewidth for large macromolecules such as cytochromes is complicated by the presence of paramagnetic species, non-spherical molecules and protein mobility. However, for haem C12 methyl the linewidth, in the diamag- netic state, is estimated to be 10- 14 Hz and it was assumed that the 10-Hz difference between the linewidth of the complexed and free cytochrome b5 is indicative of protein association. Similar reasoning to the above accounts for a linewidth of approximately 68 Hz for a trimeric cytochrome c/b5 complex.

It is clear that the dissociation of the binary complex to monomeric cytochrome c and b5 at higher ionic strengths

occurs in a stepwise manner. This observation implies that the dynamics of the interaction between cytochromes c and b5 are modulated by ionic strength and that at least two distinct states exist for the binary complex which differ in their dynamic properties and stability. The decreased association of cytochrome bs with cytochrome c at higher ionic strengths is not surprising in view of the number of potential ion-binding domains on the surface of each protein. The association of electrolyte with the complex weakens the electrostatic contacts between the proteins as a result of shielding charges on the surface of each redox partner. Of more interest is the observation that the interaction of electrolyte with the surface of the binary complex produces a stepwise decrease in rotational mobility. This leads to at least two states for the complex in which the proteins have different molecular motions that can be ‘fine-tuned’ by the ionic medium. It should therefore be noted that the modulation of intra-complex mobility by electrolyte would make the first-order electron transfer rate dependent on ionic strength but as yet such data have not been reported.

The results obtained here have considerable relevance to possible modes of long-range electron transfer between proteins which will be discussed in a subsequent paper elsewhere [49]. It is probable that the second cytochrome c molecule binds either directly to cytochrome b5 or is in contact with both proteins of the binary complex and can influence electron transfer kinetics directly or indirectly. The potential modulation of electron transfer kinetics at the primary site through the regulation of intra-complex mobility is inferred by the result obtained here. These possibilities provide numerous complications in interpreting rates of intra-complex electron transfer although the relative affinities of the two sites are such that for equimolar mixtures of the proteins the effects of secondary binding are likely to be minimal.

To date descriptions of the interaction of protein partners have centred largely on the juxtaposition of fixed static surface charges. In view of the results obtained here and current ideas on protein motions, this is clearly an inaccurate model. In attempting to overcome these limitations picosecond simulations of the movement of protein partners with the cytochrome b5/c complex have highlighted the movement of the invariant Phe82 of cytochrome c into a position at the interface between the binary complex. NMR parameters (i.e. chemical shifts) are sensitive to kinetic fluctuations on the lo4 s-’ time scale that are often characteristic of dynamic processes occurring in most protein-mediated electron transfer reactions. The absence of major chemical shift changes in the aromatic or aliphatic regions for amino acids known to constitute the interaction domain argues against any conformational changes in the binary complex. It was further con- cluded, in view of the insensitivity of the chemical shift of the Phe82 ortholmeta resonance upon binding cytochrome bs, that kinetic dislocation of the aromatic ring at the protein interface does not occur within the time range monitored by NMR. It seems therefore unlikely that picosecond fluctuations of the aromatic ring of Phe82 are significant to electron transfer reactions. Furthermore it has been shown that the involvement of aromatic residues directly in electron transfer is only likely to be important in reactions of high thermodynamic driving force [50, 511. The driving force for the cytochrome b5/c couple is estimated to be 200 -250 mV so it seem unlikely that Phe82 plays a direct role in this intracomplex electron transfer [52, 531.

In conclusion, it seems unlikely that molecular recognition of cytochrome c by cytochrome bs and the rate of subsequent

electron transfer could be interpreted solely in terms of a static binary 1 : 1 model. The present results raise the possibility that electron transfer between cytochrome c and cytochrome bs occurs in protein - protein configurations that differ from the ground state complex.

This work has been supported from grants provided by the Sci- ence and Engineering Research Council and the Nuffeld Foundation.

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