Weak interactions and molecular recognition in systems involving electron transfer proteins

T H E C H E M I C A L R E C O R D

290 © 2001 The Japan Chemical Journal Forum and John Wiley & Sons, Inc.

T H EC H E M I C A L

R E C O R D

Weak Interactions and MolecularRecognition in Systems Involving ElectronTransfer Proteins

SHUN HIROTA,1 OSAMU YAMAUCHI2

1Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku,Nagoya 464-8602, Japan

2Unit of Chemistry, Faculty of Engineering, Kansai University, Yamate-cho, Suita,Osaka 564-8680, Japan

Received 19 October 2000; accepted 15 November 2000

ABSTRACT: Electrostatic interactions and other weak interactions between amino acid side chainson protein surfaces play important roles in molecular recognition, and the mechanism of their inter-molecular interactions has gained much interest. We established that charged peptides are useful forinvestigating the molecular recognition character of proteins and their molecular interaction inducedstructural changes. Positively charged lysine peptides competitively inhibited electron transfer fromreduced cytochrome f (cyt f) or cytochrome c (cyt c) to oxidized plastocyanin (PC), due to neutraliza-tion of the negatively charged site of PC by formation of PC–lysine peptide complexes. Lysine peptidesalso inhibited electron transfer from cyt c to cytochrome c peroxidase. Likewise, negatively chargedaspartic acid peptides interacted with the positively charged sites of cyt f and cyt c, and competitivelyinhibited electron transfer from reduced cyt f or cyt c to oxidized PC and from [Fe(CN)6]

4– to oxidizedcyt c. Changes in the geometry and a shift to a higher redox potential of the active site Cu of PC onoligolysine binding were detected by spectroscopic and electrochemical measurements, owing to theabsence of absorption in the visible region for lysine peptides. Structural and redox potential changeswere also observed for cyt f and cyt c by interaction with aspartic acid peptides. ©2001 The JapanChemical Journal Forum and John Wiley & Sons, Inc. Chem Rec 1:290–299, 2001

Key words: electron transfer protein; charged peptide; weak interaction; molecular recognition;protein–peptide complex

Intermolecular Interaction forElectron Transfer Proteins

v Correspondence to: S. Hirota (E-mail: [email protected]);O. Yamauchi (E-mail: [email protected]).

Contract grant sponsor: Ministry of Education, Culture, Sports, Scienceand Technology of Japan.

Proteins, including electron transfer proteins, recognize theirpartners in specific ways, which is very important for their func-tion. Weak interactions, such as electrostatic and hydrophobicinteractions between amino acid residues on the surfaces of pro-teins, play important roles in molecular recognition. Due to theimportance of these interactions for recognition, the mechanismof interactions between proteins has gained much interest. Wehave recently shown that charged peptides are useful for investi-gating the molecular recognition character of proteins and their

interaction induced structural changes. In this short account,we discuss our method for investigating the intermolecular elec-trostatic interaction between electron transfer proteins.

The Chemical Record, Vol. 1, 290–299 (2001)© 2001 The Japan Chemical Journal Forum and John Wiley & Sons, Inc.

We a k I n t e r a c t i o n s a n d M o l e c u l a r R e c o g n i t i o n

© 2001 The Japan Chemical Journal Forum and John Wiley & Sons, Inc. 291

Many electron transfer proteins are charged on their sur-faces and/or have specific charged sites for complex formationwith their partners. For these proteins, the electrostatic inter-action is one of the main forces that guide them to specificcomplex formation. For example, cytochrome c (cyt c) is a wellknown electron transfer heme protein that is positively chargedat neutral pH,1,2 whereas one of its electron transfer partners,cytochrome c peroxidase (CcP), is negatively charged at neu-tral pH.3 The cyt c–CcP complex is thus formed by the electro-static interaction, which has been verified by X-ray crystalstructure analysis.4 The cytochrome b6f complex is an integraloligomeric membrane protein complex existing in photosyn-thetic organisms, where cytochrome f (cyt f ) donates electronsto a copper protein, plastocyanin (PC). A lysine residue-richpositive patch has been revealed by the X-ray structural studiesto exist at a solvent-exposed site of chloroplast cyt f .5 PC is amobile electron transfer protein existing in the thylakoid lu-men, and it is negatively charged at neutral pH. In PC, a site

containing consecutive acidic residues (negative patch) is lo-cated at a solvent-accessible site near a tyrosine residue remotefrom the Cu center.6,7 The electrostatic interactions betweenthe positive patch of cyt f and the negative patch of PC havebeen predicted to be the force for formation of the cyt f–PCcomplex (Fig. 1), similar to that for formation of the cyt c–CcPcomplex. Increase of the Soret band intensity of cyt f or cyt c onbinding of PC has been noted, and the association constants forbinding of cyt f or cyt c with PC have been obtained.8–10

An NMR study on the Cd-substituted PC–cyt c complexindicated that the PC–cyt c complex consists of a highly dy-namic ensemble of structures,11 whereas three docked complexesof PC and cyt f were shown by a dynamic analysis of the elec-trostatics.12 Recently, the effects of binding of cyt f on the back-bone and side-chain protons of PC have been analyzed bymapping NMR chemical-shift changes.13 Protons with signifi-cant chemical-shift changes were located in both the hydro-phobic and negative patches of PC, demonstrating that both

v Osamu Yamauchi was born in Nagoya in 1936. He received his master’s degree from KyotoUniversity in 1961. After spending 2 years at a pharmaceutical company as a research staffmember, he resumed his graduate studies at Kyoto University, receiving his doctor’s degree inpharmaceutical sciences in 1967. He was appointed Associate Professor at Osaka University in1967 and worked on copper-peptide chemistry with Professor Akitsugu Nakahara. During theperiod 1971–73 he worked with Professor Jui H. Wang at Yale University and then at the StateUniversity of New York at Buffalo on oxidative phosphorylation. He became full Professor atKanazawa University in 1980, and in 1987 he was appointed Professor in the Department ofChemistry, Nagoya University. He retired from Nagoya University in 2000 and is now Professorat Kansai University and Professor Emeritus of Nagoya University. His research interests includeweak interactions involving metal-coordinated amino acids and metalloproteins, self-organiza-tion of complex molecules, reactivity of aromatic rings in the vicinity of the metal center, metal-pterin chemistry, and other areas of bioinorganic chemistry. r

v Shun Hirota was born in Port Jefferson, New York, in 1965. He obtained his Bachelor ofEngineering and Master of Engineering degrees from Kyoto University. He received his PhDdegree in 1995 from the Graduate University for Advanced Studies under the supervision ofProfessor Teizo Kitagawa at the Institute for Molecular Science in Okazaki. He was a JSPSResearch Fellow from 1994 to 1996 and joined Professor L. G. Marzilli’s research group in theDepartment of Chemistry, Emory University, in 1995. Since 1996, he has been an AssistantProfessor in the Department of Chemistry, Graduate School of Science, Nagoya University. Hereceived The Chemical Society of Japan Award for Young Chemists for 2000. He has beenworking in the field of bioinorganic chemistry, and his research interests include structure-func-tion relationship and reaction mechanisms of metalloproteins. r



areas are part of the interface in the PC–cyt f complex. Thelargest chemical-shift changes were found around His87 in thehydrophobic patch, which indicated tight contact and possi-bly water exclusion from this part of the protein interface. Theseresults supported the idea that electron transfer occurs via His87to the copper in PC.13 Theoretical docking models also sug-gested that the negative patch of PC is not important in thecomplex that is active in electron transfer, although it is impor-tant for the initial binding.12,14 It has been proposed that PCand cyt f or cyt c bind and react with each other in differentconfigurations resulting from the protein-protein interaction,termed as the gating process for electron transfer.15–19 Paramag-netic NMR and restrained rigid-body molecular mechanicsstudies indicated that the electrostatic interactions guide PCand cyt f into a position that is optimal for electron transfer.20

However, kinetic evidence for multiple binary complexes hasbeen shown for the protein docking and gated electron trans-fer reactions between zinc cyt c and the new PC from the fern

Dryopteris crassirhizoma,21 which has a very large acidic surfaceextending into the area that is hydrophobic in other PCs.22

Interaction of Electron Transfer Proteins withInorganic Compounds

Inorganic compounds have been used extensively for studieson the molecular interaction character of proteins. For example,electron transfer kinetic measurements between blue copperproteins and optically active FeII and CoII complexes have beendone as a function of temperature and pH, which showed thatlow molecular weight electron transfer reagents can react atdifferent sites of a narrow area of the protein surface.23 Elec-tron transfer between PC and inorganic complexes has beenreported,24–26 and two binding sites, Cu-adjacent hydrophobicand Cu-remote negative patches, have been demonstrated forthe oxidation of PC with inorganic complexes.25,27,28 Both the

Fig. 1. Schematic view of the interaction between cyt f and PC in the presence of a lysine peptide (PDB 1CTM and 1BYO).



hydrophobic and negative patches of PC have also been sug-gested to be the molecular recognition sites for cyt f by studieson electron transfer from reduced PC to [Co(phen)3]

3+ (phen =1,10-phenanthroline).27,28

Redox-inactive inorganic compounds have also been em-ployed for studying the molecular recognition character of elec-tron transfer proteins. Sykes et al. showed that small redox-inactiveinorganic compounds inhibit electron transfer between PC andcyt c,29 cyt f,30 or inorganic redox partners,28,31,32 which was ex-plained by competitive inhibition due to interaction betweenthe positive charge of the inorganic compounds and the nega-tive charge of PC.

NMR investigations on the complex formation betweenproteins and CrIII compounds have been made,33,34 and boththe hydrophobic and negative patches were shown to be the[Cr(NH3)6]

3+ interacting sites for PC,34 whereas several[Cr(NH3)6]

3+ binding sites were detected for cyt c.33 Williamsand coworkers identified eight ion-binding sites on the surfaceof cyt c by the NMR paramagnetic difference spectrummethod.35–37

Effect of Charged Peptides on ElectronTransfer Proteins

Effect on Electron Transfer Reaction between Proteins

The electrostatic interaction is essential for complex formationin many electron transfer proteins. To study the intermolecu-lar interaction character of proteins, we have been using chargedoligopeptides as models for the protein interacting sites. Posi-tively charged peptides would interact with the aspartic acid

and glutamic acid residues of the negatively charged sites ofproteins, whereas the negatively charged peptides can interactwith the lysine residues of the positively charged sites. Actu-ally, poly-L-lysine has been shown to interact with proteins.For example, poly-L-lysine inhibited the oxidation of eukary-otic cyt c by eukaryotic cytochrome c oxidase,38 while it stimu-lated oxidation of Paracoccus denitrificans cyt c by P. denitrificansoxidase.39,40 Stimulation of cholesterol binding to steroid-freecytochrome P-450scc by poly-L-lysine has also been mentioned.41

However, it is difficult to achieve quantitative and detailed stud-ies using poly-L-lysines.

More detailed studies could be performed with the use ofcharged oligopeptides. Positively charged lysine (Lys) peptidesinteracted with the negative patch of PC and inhibited elec-tron transfer from reduced cyt f or cyt c to oxidized PC (Figs.1, 2).42,43 Addition of NaCl to the solution was necessary tomake the electron transfer rate detectable by stopped-flowmeasurements, and a higher NaCl concentration was neededfor the PC–cyt f system than for PC–cyt c because the associa-tion was stronger between PC and its native redox partner, cytf, than between PC and cyt c. The inhibition of electron trans-fer became prominent as the length and concentration of theLys peptide increased (Fig. 2), but no inhibition was observedwhen the same amount of a glycine (Gly) peptide, tetra-Gly,was added, showing that the effect of the peptide terminalcharges of the –COO– and –NH3

+ groups can be neglected.The inhibition was rather weak when shorter Lys peptides (tri,di, or mono) were used, due to their weaker electrostatic inter-actions with PC. The electron transfer rate from reduced cyt cto oxidized PC and the inhibitory effect of Lys peptides de-creased upon decreasing the net charge of the negative patch

Fig. 2. Plots of the reciprocal electron transfer rate constants (1/kobs) from reduced cyt f or cyt c to oxidized PCversus the initial concentrations of Lys peptides: di- (X), tri- (Z), tetra- (5), and penta- (2) Lys. (A) Reduced cytf–oxidized PC and (B) reduced cyt c–oxidized PC electron transfer systems. Tris-HCl buffer (10 mM), pH 7.3,containing (A) 10 mM and (B) 60 mM NaCl was used. Modified from references 42 and 43.



by mutation.42 Taken together, these effects established thatLys peptides having positive charges interact with the asparticacid and glutamic acid residues of the negative patch of PCand decrease the electron transfer rate. The inhibitory effectsof Lys peptides on electron transfer were thus explained as com-petitive inhibition due to neutralization of the PC negative patchby formation of PC-Lys peptide complexes (Fig. 1). The re-sults strongly support that the PC negative patch is importantfor complex formation between PC and cyt f/c.

The observed inhibition by Lys peptides in the cyt f/c–PCsystem may be interpreted by considering formation of twocomplexes: a cyt f/c–PC complex where electron transfer oc-curs subsequently, and a PC–Lys peptide complex, which com-petitively inhibits formation of the PC–cyt f/c complex andthus inhibits electron transfer. Since it is strongly supportedthat the negative patch is the dominant protein recognitionsite,42 we assumed that Lys peptide binds effectively only at thePC negative patch. The complex formations are expressed bythe following equations:

→+ − →←−

K k f/c f/c

f/c

os eox red ox red R

red ox R

PC cyt (PC cyt )

(PC cyt )(1)

→+ −←K i

ox ox RPC Lys peptide (PC Lys peptide) (2)

where KOS and Ki are the association constants for PCox–cytf/cred and PCox–Lys peptide complexes, respectively, and ke rep-resents the electron transfer rate constant. The suffixes ox andred refer to the oxidized and reduced states, respectively, and Rdenotes that the complex is formed at the Cu-remote negative

patch of PC. If we write the observed rate constant as kobs, KOS

× ke as k, and the initial concentrations of PC and lysine pep-tide as [PC]0 and [Lys peptide]0, respectively, we obtain thefollowing relationship when [PC] >> [cyt f/c]:

= × +× ×

K

k k ki

0obs 0 0

1 1[Lys peptide]

[PC] [PC] (3)

Plots of 1/kobs versus [Lys peptide]0 gave straight lines, and kand the association constant Ki values for the PC–Lys peptidecomplexes were obtained for both the PC–cyt f and PC–cyt csystems from least-squares fitting of the data with Equation 3[Figs. 2, 3(A) and Tables 1 and 2]. The Ki values decreasedsignificantly at higher NaCl concentrations of the solution,which supported that the interaction between PC and the Lyspeptide is electrostatic. Because the fitting was successful, it isreasonable to assume that only one Lys peptide is bound to thenegative patch of PC. Likewise, tetra- and penta-Lys served ascompetitive inhibitors of electron transfer from reduced cyt cto CcP [CcPIV=O(Trp191•,+)],44 which was ascribable to forma-tion of similar electrostatic CcP–Lys peptide complexes.

Aspartic acid (Asp) peptides also served as competitiveinhibitors of electron transfer from reduced cyt f or cyt c tooxidized PC (Fig. 3).43 As shown in the following equation

= × +× ×K ´

k k ki

0obs 0 0

1 1[Asp peptide]

[PC] [PC] (4)

a similar relationship is obtained for the inhibitory characterof Asp peptides in the cyt f –PC electron transfer system, where

Fig. 3. Plots of the reciprocal electron transfer rate constants (1/kobs) from reduced cyt f or cyt c to oxidized PCversus the initial concentrations of Asp peptides: di- (X), tri- (Z), tetra- (5), and penta- (2) Asp. (A) Reduced cytf –oxidized PC and (B) reduced cyt c–oxidized PC electron transfer systems. Tris-HCl buffer (10 mM), pH 7.3,containing (A) 10 mM and (B) 60 mM NaCl was used. Modified from reference 43.



Ki′ is the association constant for cyt fred-Asp peptide and [as-partic acid peptide]0 is the initial concentration of the Asp pep-tide. Ki′ values for cyt f and tetra- and penta-Asp are listed inTable 1.43 The association constants Ki and Ki′ obtained underthe same condition were larger for the penta peptide comparedto the tetra peptide, and the constants obtained for cyt f–Asppeptide and PC–Lys peptide complexes with the same peptidelength showed similar values (i.e., cyt f–tetra-Asp vs PC–tetra-Lys and cyt f–penta-Asp vs PC–penta-Lys). Therefore, cyt f andPC would interact with Asp and Lys peptides, respectively, withcomparable electrostatic strength.

For the cyt c–PC system, inhibition by Asp peptides didnot follow the equations discussed above (Eq. 1–4) [Fig. 3(B)].The inhibition was not significant when the concentration ofthe peptides was low, so that higher concentrations were re-quired for effective inhibition. The difference in the inhibi-tory character of Lys peptides and Asp peptides in the cytc–PC system may be due to the difference in how cyt c andPC recognize their reaction partners, because the distribu-tion of the charged amino acids on the surface of each pro-tein is different. An aforementioned NMR study on the cytc–PC complex showed that the negative patch of PC is theinteraction site with cyt c, while a large area around the hemeedge of cyt c is involved in the interaction with PC.11,20 Asdiscussed in the section “Protein Structural Change Inducedby Interaction with Charged Peptides,” the redox potentialof cyt c slightly shifts to a lower potential on Asp peptidebinding, and the peptide bound at one site of cyt c could

increase the electron transfer rate from cyt c to PC throughthe other peptide binding sites of cyt c, because there are sev-eral peptide and PC binding sites on the surface of cyt c. Thus,the inhibitory effect of Asp peptides on electron transfer mightnot be effective at low peptide concentrations, due to cancel-lation between this promoting effect by the shift in the redoxpotential and the inhibitory effect by the peptides bound tothe electron transfer sites of the protein. The inhibition couldoccur only when the positive charges around the heme edgeare considerably blocked by the peptides due to formation ofcyt c–(Asp peptide)n complexes (n > 1), which should requirea certain concentration of the peptide. It is interesting thatthe inhibitory effect dependence on peptide concentrationvaried among proteins according to their molecular interac-tion character.

Effect on Electron Transfer Reaction Involving[Fe(CN)6]

4–

Interactions of charged oligopeptides with cyt c or PC werealso studied by measuring electron transfer between [Fe(CN)6]

4–

and oxidized cyt c or PC in the presence of peptides.45 Asppeptides up to penta-Asp served as competitive inhibitors ofelectron transfer from [Fe(CN)6]

4– to oxidized cyt c, while noinhibition was observed in the same electron transfer reactionwhen the same amount of tetra-Gly was used. As in the cyt f/c–PC system, the observed inhibition of the [Fe(CN)6]

4––cyt celectron transfer reaction by Asp peptides may be interpretedby considering two kinds of interactions: a cyt c–[Fe(CN)6]

4–

interaction, where electron transfer occurs subsequently, and acyt c–Asp peptide interaction, which competitively inhibits for-mation of the cyt c–[Fe(CN)6]

4– complex at an [Fe(CN)6]4– in-

teracting site of cyt c, and thus decreases the electron transferrate. We assumed that there are n sites for cyt c to interact with[Fe(CN)6]

4– or the Asp peptide. We defined Kin and KOSn

(n =1, 2, ··· , n) as the association constants for cyt cox–Asp peptideand cyt cox–[Fe(CN)6]

4– complexes at site n, respectively, andken

as the electron transfer rate constant through this site. Forsimplicity, we assumed that the Asp peptide bound on one siteof cyt c does not affect the association constant or the electrontransfer rate constant between cyt c and [Fe(CN)6]

4– at othersites, and that Kin

, KOSn, and ken

are the same (Kin = K, KOSn

=KOS, and ken

= ke ) for all cyt c sites with which [Fe(CN)6]4– or

the Asp peptide interacts. However, only certain sites of cyt cwith relatively large association constants for [Fe(CN)6]

4– andlarge electron transfer rates should be responsible for electrontransfer. If we write the observed rate constant as kobs, KOS × ke

as k′, and the initial concentration of cyt c, [Fe(CN)6]4–, and

Asp peptide as [cyt c]0, [[Fe(CN)6]4–]0, and [Asp peptide]0, re-

spectively, we obtain the following relationship when[[Fe(CN)6]

4–]0 >> [cyt c]0:

Table 1. Association constants (Ki and Ki′) for PC–Lys peptide andcyt f–Asp peptide complexes [a].

Association constant/M–1

Length of peptide PC–Lys peptide (Ki) cyt f–Asp peptide (Ki′)

tetra (8.2 ± 0.5) × 102 (7.60 ± 0.6) × 102

penta (1.3 ± 0.1) × 103 (1.0 ± 0.1) × 103

[a]Obtained by least-squares fitting of the data in Figures 2(A) and 3(A) withEquation 3, in 10 mM Tris-HCI buffer, pH 7.3, containing 60 mM NaCl, at15°C.

Table 2. Association constants (Ki) for PC–Lys peptide complexes [a].

Length of Lys peptide Ki/M–1

di (5.5 ± 1.3) × 102

tri (1.4 ± 0.2) × 103

tetra (5.9 ± 0.3) × 103

penta (15.3 ± 1.0) × 103

[a]Obtained by least-squares fitting of the data in Figure 2(B) with Equation3, in 10 mM Tris-HCl buffer, pH 7.3, containing 10mM NaCl at 15°C.



−−−

−−−

+ − +

− +

+×

+ +

+

+

1 0

222 0

111 0

obs 1 0

222 0

111 0

( 1) C [Asp peptide]

( 2) C [Asp peptide]

C [Asp peptide]= 1 C [Asp peptide]

C [Asp peptide]

C [Asp peptide]

n

n

nnn n

n

n

nnn n

n n K

n K

... Kk k

K

K

... K

(5)

where n denotes the number of bound Asp peptides.Electron transfer rate constants for several singly modi-

fied cyt c derivatives, where specific Lys residues at the exposedheme edge were modified to neutral or negatively charged spe-cies, decreased from the constant for native cyt c.46,47 Least-squares fitting of kobs was successful only for n ≥ 6, supportingthat cyt c has many sites for [Fe(CN)6]

4– interaction. Actually,cyt c recognizes its redox partner protein through more thantwo of its positively charged sites, and four anion binding siteshave been identified by NMR spectroscopy.35–37 k′ was calcu-lated to be 128 ± 5 s–1 when n = 6, and the K values for variousAsp peptides obtained for n = 6 are listed in Table 3. K in-creased as the length of Asp peptide increased, which is in agree-ment with the interpretation that Asp peptide electrostaticallyinteracts with the positively charged amino acid residues onthe surface of cyt c.

Lys peptides up to penta-Lys promoted electron transfer from[Fe(CN)6]

4– to oxidized PC. It has been suggested that inorganiccomplexes bind with PC at both Cu-adjacent and Cu-remotesites.28,31,32 The electron transfer promoting effects of Lys pep-tides may be due to formation of PC–Lys peptide or Lys pep-tide–[Fe(CN)6]

4– complexes, subsequently forming an electrontransferring complex, PC–Lys peptide–[Fe(CN)6]

4–, without re-pulsion of the negative charges. Binding of positively chargedLys peptides to the negative patch of PC decreases its net nega-tive charge and would facilitate electron transfer through thenegative patch, since both [Fe(CN)6]

4– and the negative patchare negatively charged. The electron transfer rate increased andthe inhibitory effect of Lys peptides decreased as the net chargeof the PC negative patch was decreased by mutagenesis, because

the repulsive interaction of the negative patch with [Fe(CN)6]4–

and the attractive interaction with the Lys peptide decrease bydecreasing the net charge of the patch. The PC negative patchthus became the dominant binding site for [Fe(CN)6]

4– whenthe concentration and charge of the Lys peptide were large, andthe net charge of the negative patch was small.

Protein Structural Change Induced byInteraction with Charged Peptides

A strong advantage of using peptides as models of the interact-ing proteins is that they do not have any visible absorption,which makes them suitable for investigating the molecular in-teraction induced structural changes of metalloproteins hav-ing absorption bands in the visible region. Effects of polyanionson the structure48–50 and redox properties51 of cyt c have beenstudied. We observed structural changes for PC and CcP uponinteraction with Lys peptides and for cyt f and cyt c upon inter-action with Asp peptides.42–44,52,53 In this section, we focus onthe use of Lys peptides to study the structural change of PC bycomplex formation (Fig. 4).

PC is a type 1 copper protein containing one copper atomwith two histidine nitrogen atoms, one methionine sulfur atom,and one cysteine (Cys) sulfur atom coordinated in a distortedtetrahedral geometry.6,7,54 An intense absorption band at about600 nm in the absorption spectrum of PC has been assigned tothe Cys thiolate [S(Cys)]-to-Cu(II) charge transfer (CT)band.55,56 In the difference absorption spectrum of PC betweenthe spectra with and without penta-Lys, peaks and troughs weredetected at about 460 and 630 nm and at about 560 and 700nm, respectively (Fig. 5). These changes revealed that the 600-

Table 3. Association constants (K) for cyt c–Asp acid peptidecomplexes [a].

Length of Asp acid peptide K/M–1

di (2.4 ± 0.5) × 102

tri (6.3 ± 1.0) × 102

tetra (1.9 ± 0.2) × 103

penta (4.6 ± 0.5) × 103

[a]In 10 mM Tris-HCl buffer, pH 7.3, containing 10 mM NaCl, at 15°C.Fig. 4. A schematic view of silene PC (PDB 1BYO) interacting withtetra-Lys.



nm absorption band shifted to a longer wavelength and thatthe 460- and 700-nm absorption bands increased and decreasedin their intensities, respectively, on interaction with Lys pep-tides, which indicates that the structure of the Cu site [at leastthe Cu-S(Cys) bond] should be altered by addition of Lys pep-tides (Fig. 5). That the observed intensity changes are due toformation of a 1:1 PC-Lys peptide complex was concluded fromthe dependence of the difference spectral intensity on Lys pep-tide concentration.52 The intensities of the difference peaksobserved in the difference spectra decreased when the amountof NaCl added to the solution was increased,53 which showsthat the structural change at the Cu site of PC is caused by anelectrostatic interaction of the PC negative patch with Lys pep-tide. In fact, the spectral changes of PC observed on interac-tion with Lys peptide decreased when the charges at the negativepatch were decreased by site-directed mutagenesis.42

The intensity increase of the 460-nm absorption band intype 1 copper proteins has been explained by the structuralchange from a trigonal planar toward a more tetrahedral Cusite geometry associated with the lengthening of the Cu-S(Cys)bond.57–59 The ratio of the intensity of the 460-nm absorptionband to that of the 600-nm band has been reported to corre-late with the rhombicity of the EPR signal for type 1 copper

proteins,59 and the rhombic distortion has been suggested tobe associated with a stronger binding of the axial ligand and aconcomitant shift from a trigonal planar toward a more tetra-hedral Cu site geometry.57–59 Because the intensity of the 460-nm absorption band increased and the 600-nm absorption peakshifted to a longer wavelength when PC interacted with theLys peptide, it is suggested that the Cu-S(Cys) bond was elon-gated on interaction with Lys peptide.

The CD spectrum of PC exhibited positive bands at about420, 560, and 610 nm, and negative bands at about 470 and760 nm. A positive peak at 420 nm and a negative peak at 470nm were detected in the CD difference spectrum on interac-tion with tetra-Lys, which supports changes in the active sitestructure. The 420- and 470-nm bands have been assigned tothe Met → Cu 3dx2-y2 and His π1 → Cu 3dx2-y2 CT transitions,respectively.55 In cucumber basic protein, the intensities of thesebands are stronger,60 the Cu–S(Cys) and Cu–S(Met) bondlengths are elongated and shortened by 0.1 and 0.2 Å, respec-tively,61 and the Cu–S(Cys) stretching frequency is lower,62 ascompared with the corresponding values for PC. Therefore, itis inferred that the Cu–S(Cys) and Cu–S(Met) bond lengths ofPC become longer and shorter, respectively, by interaction withLys peptides.

It has been reported that the Cu–S(Cys) stretching (ν)Cu-S

frequency is a sensitive probe of the Cu–S(Cys) bond strengthand copper coordination geometry for proteins with Cys thiolatecoordination.63–66 Several bands were observed at 374–475 cm–1

for the resonance Raman (RR) spectra in the 200–600 cm–1

region of PC excited at its S(Cys)-to-Cu(II) CT band, which ismainly due to the mixing of the Cu-S stretch with several anglebending modes of the coordinated Cys side chain.63,67–70 Whentetra-Lys was added to the PC solutions, the Raman bands at375–475 cm–1 slightly shifted to lower frequencies and the in-tensities of some lower frequency bands in this region in-creased slightly, whereas the 267-cm–1 band, which is assignedto the Cu-His stretching mode,71 did not show any change.Especially, the νCu-S related 375- and 422-cm–1 bands were af-fected. These results indicate that the Cu–S(Cys) bond wasweakened slightly on addition of the Lys peptide to PC (Fig.6). It is interesting to note that the same RR spectral changesof PC were observed for PC–cyt c and PC–Lys peptide interac-tions, indicating that Lys peptides are excellent models for thePC recognition site of proteins.

Electrochemical measurements showed that the redox po-tential of PC shifted to a higher potential upon Lys peptidebinding (Fig. 6), suggesting that Lys peptides induce a struc-tural change in PC to adapt the copper site for facile electrontransfer. In this connection, the structural change might berelated to information in recent reports that PC and cyt c orcyt f bind and react with each other in different configurationsresulting from the protein–protein interaction termed as thegating process for electron transfer.15–19,72

Fig. 5. Absorption spectra of oxidized PC (curve a) with and (curve b) with-out penta-Lys, and (curve c) their difference spectrum (curve a – curve b)multiplied by 20. The baseline of the difference spectrum is shown as a dot-ted line. Phosphate buffer (10 mM), pH 7.4, was used. Modified from refer-ence 53.



Changes in the absorption spectrum of cyt c in the Soretregion were detected when Asp peptides were added to the cytc solution. These changes were the same as those observed whencyt c interacted with PC, indicating that Asp peptides inter-acted with cyt c in the same way as PC. The conformationalchanges of cyt c due to interaction with Asp peptides observedby RR spectroscopy were also similar to those reported for in-teraction with its native partner, cytochrome c oxidase. Theredox potentials of cyt c and cyt f shifted to lower potentialsupon peptide binding, showing that the electron donor abilityof both cyt c and cyt f is enhanced upon complex formationwith Asp peptides.

Conclusion

We have shown that charged peptides are useful for studyingthe molecular recognition character of proteins and their in-teraction induced structural changes. Positively charged Lyspeptides interacted with the consecutive Asp and glutamic acidresidues of the negative patch of PC and inhibited electrontransfer from reduced cyt f or cyt c to oxidized PC. The inhibi-tory effect of Lys peptides on electron transfer was explained ascompetitive inhibition due to neutralization of the PC nega-tive patch by formation of PC–Lys peptide complexes. Lys pep-tides also inhibited electron transfer from reduced cyt c to CcP[CcPIV=O(Trp191•,+)]. Likewise, negatively charged Asp pep-tides interacted with the positively charged sites of cyt f and cytc, and competitively inhibited electron transfer from reduced

cyt f or cyt c to oxidized PC and from [Fe(CN)6]4– to oxidized

cyt c.Owing to the fact that oligopeptides have no absorption

band in the visible region and are usually redox inactive, changesin the active site Cu–Cys bond and a shift to a higher redoxpotential on binding of Lys peptides have been detected forPC by spectroscopic and electrochemical methods. These re-sults suggest that Lys peptides induce structural changes in PCto adapt the copper site for facile electron transfer. Structuraland redox potential changes were also observed for cyt c andcyt f upon interaction with Asp peptides.

We thank the coworkers whose names are listed in thepapers cited in the references for their contribution andenthusiasm.

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Weak interactions and molecular recognition in systems involving electron transfer proteins