Bioconjugate Chem. 1992, 3, 382-3911

382

Metalloprotein Complexes for the Study of Electron-Transfer Reactions. Characterization of Diprotein Complexes Obtained by Covalent Cross-Linking of Cytochrome c and Plastocyanin with a Carbodiimidet Jian S. Zhou, Herb M. Brothers 11, John P. Neddersen, Linda M. Peerey, Therese M. Cotton, and Nenad M. Kostit' Department of Chemistry, Iowa State University, Ames, Iowa 50011. Received March 23, 1992

Cytochrome c (cyt) and zinc cytochrome c (Zncyt) are separately cross-linked to plastocyanin (pc) by the carbodiimide EDC according to a published method. The changes in the protein reduction potentials indicate the presence of approximately two amide cross-links. Chromatography of the diprotein complexes cyt/pc and Zncyt/pc on CM-52 resin yields multiple fractions, whose numbers depend on the eluent. UV-vis, EPR, CD, MCD, resonance Raman, and surface-enhanced resonance Raman spectra show that cross-linking does not significantly perturb the heme and blue copper active sites. Degrees of heme exposure show that plastocyanin covers most of the accessible heme edge in cytochrome c. Impossibility of cross-linking cytochrome c to a plastocyanin derivative whose acidic patch had been blocked by chemical modification shows that it is the acidic patch that abuts the heme edge in the covalent complex. The chromatographic fractions of the covalent diprotein complex are structurally similar to one another and to the electrostatic diprotein complex. Isoelectric points show that the fractions differ from one another in the number and distribution of N-acylurea groups, byproducts of the reaction with the carbodiimide. Cytochrome c and plastocyanin are also tethered to each other via lysine residues by N-hydroxysuccinimide diesters. Tethers, unlike direct amide bonds, allow mobility of the cross-linked molecules. Laser-flash-photolysisexperiments show that, nonetheless, the intracomplex electron-transfer reaction cyt(II)/pc(II) cyt(III)/pc(I) is undetectable in complexes of either type. Only the electrostatic diprotein complex, in which protein rearrangement from the docking configuration to the reactive configuration is unrestricted, undergoes this intracomplex reaction at a measurable rate.

-

Metalloproteins are involved in various biological oxidoreduction processes, and it is important to understand the kinetics and mechanisms of their electron-transfer reactions (11. The heme protein cytochrome c (designated cyt) and the blue copper protein plastocyanin (designated pc) are well-suited to kinetic and mechanistic studies because their structures and propertiesare known in detail. Cytochrome c has a positively charged (basic) patch near the exposed heme edge (2, 3), whereas plastocyanin has a negatively charged (acidic) patch remote from the copper atom and an electroneutral (hydrophobic) patch proximate to this atom ( 4 ) . According to quantum-chemical calculations, electron-transfer paths connecting the copper atom to the hydrophobic patch are more efficient than paths connecting this atom to the acidic patch (5). The presence in plastocyanin of two putative sites for interaction with redox partners makes this protein especially interesting. Reaction of plastocyanin with cytochrome c, and a reaction between two redox proteins in general, involves three general steps: association of the proteins, electron transfer, and dissociation of the diprotein complex (6). The second step (eq 1) can be experimentally separated + Abbreviations used: CD, circular dichroism; cyt, cytochrome c; cyt(II), ferrocytochrome c; cyt(III), ferricytochrome c; DST, disuccinimidyl tartarate; EDC, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide hydrochloride; EGS, ethylene glycol bis(succinimidyl succinate); L, flavin; LH', flavin semiquinone; MCD, magnetic circular dichroism; MOPS, 3-(N-morpholino)propanesulfonate; NHE, normal hydrogen electrode; pc, plastocyanin; pc(I),cuproplastocyanin; pc(II), cupriplastocyanin; RF, riboflavin;RFH', riboflavin semiquinone; RRS, resonance Raman spectroscopy; SDS, sodium dodecyl sulfate; SERRS, surfaceenhanced resonance Raman spectroscopy;Zncyt, zinc cytochrome C.

-

Cyt(II)/PC(II)

Cyt(III)/PC(I)

(1)

from the other two by varying ionic strengths and by crosslinking the proteins. The photoinduced "forward"reaction kf is followed by the thermal "back" reaction k b (eq 2).

ki

kb

3 ~ n c y t / p c ( ~ ~Zncyt+/pc(I) )

Zncyt/pc(II)

(2)

Besides these unimolecular reactions within diprotein complexes, there are also bimolecular reactions between diprotein complexes on the one side and various external redox agents on the other (eqs 3-9). The Roman numerals cyt(III)/pc(II)

cyt(Il)/pc(l)

+

LH'

+ Fe(II1)

-c -c-

CYt(II)/PC(II)+ pc(II)

Cyt(III)ipc(II) + cyt(I1) 3 ~ n c ~ ~+ cPCUQ ( ~ ) CYt(WPC(I0 + CyVPC(I0 3 ~ n c ~ p c+( ~ Zncyt/pc(II) )

-

-

cYt(Il)/Pc(II)

t

+ L + H+

(3)

+ Fe(I1)

(4)

cyt" Cyt(Il)/PC(II) CYt(IIWpc(l) Cyt(~IB/pc(II)+ pc(0 cyt(III)/pc(I)

+

cyt(1II)

(5) (6)

Zncyt+/pc(o + pcm

(7)

CYt(IIl)/PC(Il) + cyvpcm

(8)

zncyt+/pc(n + Zncwpc(I)

(9)

show oxidation states of iron and copper; the one oxidation state omitted in eq 8 is irrelevant. Kinetic and mechanistic studies in this laboratory of the reactions in eqs 1-9 have yielded unexpected results (7-12). The electron-transfer reaction in eq 1within the electrostatic complex (1200 s-l) is abolished (less than 0.2 s-l) when this complex is reinforced by noninvasive crosslinks, promoted by the carbodiimide EDC, which prevent 0 1992 American Chemical Society

Protein Crosslinking with Carbodiimlde

protein rearrangement. But the reactions kfand k b in eq 2 within the two covalent complexes still occur (kf= 2.2 X lo4and kb = 7 X lo4 s-l, respectively) despite identical cross-linking. This remarkable contrast can be explained on assumption that the amide cross-links connect residues near the heme edge to those in the acidic patch. Native cytochrome c, a weak reductant (E" = 0.26 V vs NHE), apparently cannot reduce cupriplastocyanin (E" = 0.36 V vs NHE) from the acidic patch, which is remote from the copper atom. The triplet state of zinc cytochrome c, a strong reductant (E" = -0.88 V vs NHE) (13),apparently can reduce cupriplastocyanin from the acidic patch. Since the native and zinc-substituted cytochrome c have similar conformations, topographies, and charge distributions, protein-protein orientation for electron transfer apparently depends on the driving force for this reaction. Reactivity of the cross-linked proteins toward flavin semiquinones, designated LH' (eq 3), and toward ferric complexes, designated Fe(II1) (eq 4), cannot be predicted simply on the basis of the reactivity of separate proteins. The surprisingly small steric shielding of the iron and copper sites in the covalent complex may indicate that the proteins have multiple reaction domains on their surfaces, or that the complex is dynamical, or both. This shielding is greater in the reactions between diprotein complexes and single proteins (eqs 5-7), and even greater in the reactions between diprotein complexes (eqs 8 and 9). The reactions in eqs 1,2, and 5-9 perhaps are relevant to processes involving oligomeric redox enzymes. Our analyses of the kinetic results and the aforementioned conclusions rested on suppositions and indirect evidence about the configuration of cytochrome c and plastocyanin in the covalent complex cytfpc and about the causes of heterogeneity of this complex when it is subjected to cation-exchange chromatography. Direct evidence, presented here, confirms previous hypotheses and validates previous conclusions. Carbodiimides are routinely used for cross-linking of proteins, but the resulting complexes and aggregates are often studied in situ, without isolation. In this study, however, purification and characterization of diprotein complexes may show the pitfalls of cross-linking. Besides this analytical part, this report has a kinetic part. Cytochrome c and plastocyanin are cross-linked by bifunctional N-hydroxysuccinimide diesters. These flexible diprotein complexes, which contain tethers, are compared with rigid complexes, which contain direct amide bonds, in their (un)reactivity with respect to intracomplex electron transfer (eq 1). EXPERIMENTAL PROCEDURES

Chemicals. Chromatographic supplies, riboflavin, protein standards for determination of molecular mass, EDC, and MOPS were obtained from Sigma Chemical Co., the laser dye LD-425 was from Exciton Co., and DST and EGS were from Pierce Chemical Co. Other chemicals were of the reagent grade, and water was demineralized to a resistivity greater than 10 M a cm. All the phosphate buffers had a pH of 7.0, and their concentrations or ionic strengths are specified. Protein Purification, Reconstitution, and Modification. Horse heart ferricytochrome c (type I11 from Sigma Chemical Co.) was purified by cation-exchange chromatography (14). The free-base form of this protein was prepared, purified, and reconstituted with zinc by standard procedures, in the dark (15,16). Zinc cytochrome c was always handled in the dark. French bean plastocyanin was isolated by standard methods (17) and purified

Biomnjugete Chem., Voi. 3, No. 5, 1992 383

repeatedly by gel-filtration chromatography on Sephadex G-25 and G-75 columns with 50 mM acetate buffer at pH 6.0 and by anion-exchange chromatography on a Sephadex DEAE A-25 column with the same buffer, which was made 0.200 M in NaC1. The purifications continued until the absorbance quotient A278/A597 became less than 1.20. The acidic patch was chemically modified by ethylenediamine by a published method (18);the chromatographic fraction containing four to six blocked carboxylic groups (19,201 was used in further experiments. Ultrafiltration was done in Amicon cells, with YM-5 membranes, at 4 "C, under pressure of pure nitrogen. Protein Cross-Linking with EDC. The covalent complex cytfpc was prepared by an extension (7) of the published method (21). The product was purified on a column of Sephadex G-75 (50 mesh) sized 1.0 X 75 cm, with an 85 mM phosphate buffer a t pH 7.0 as an eluent. The major band (70-go%), containing the covalent complex cytfpc, was followed by two minor bands, containing the separate proteins. The complex (37 mg or 1.6 pmol) was dialyzed by ultrafiltration into a 5 or 10 mM phosphate buffer at pH 7.0, treated with a small excess of &[Fe(CN)61 dissolved in this buffer, concentrated, and chromatographed on a CM-52 column sized 2.5 X 8.5 or 1.5 X 20 cm that had previously been equilibrated first with a concentrated and then witha 5 or 10mMphosphate buffer at pH 7.0. Elution with a 5 mM phosphate buffer at pH 7.0 yielded 12 bands, but only the first, second, fourth, and seventh were major. Elution with a 10 mM phosphate buffer at pH 7.0 yielded four bands, and the fourth one required a shallow gradient from a 10to a 30 mM buffer. The four major bands in the former procedure correspond in their mobility to the four bands in the latter procedure. Subsequent experiments were done with the latter. The average chromatographic yields of these bands from several syntheses were 19,26, 29, and 26% in the order of elution. In another cross-linking procedure, native plastocyanin was replaced by a derivative whose acidic patch had been blocked by ethylenediamine (seethe preceding subsection). The mixture after incubation was chromatographed on a CM-52 column sized 2.5 X 15cm. Two plastocyanin bands were eluted with a 20 mM phosphate buffer at pH 7.0; two cytochrome c bands and two more plastocyanin bands were eluted with an 85 mM phosphate buffer at pH 7.0. Each fraction was oxidized with a small excess of dissolved &[Fe(CN)6] and examined by UV-vis spectrophotometry. In yet another cross-linking procedure native cytochrome c was replaced by zinc cytochrome c, and larger quantities of the proteins were used 132 mg (10.6 pmol) of zinc cytochrome c and 110 mg (10.6 pmol) of plastocyanin. Experiments were done in the dark as often as possible. Photodegradation of zinc cytochrome c was not detected. The Zncyt/pc(II) complexeluted from Sephadex G-75 was dialyzed into a 5 mM phosphate buffer at pH 7.0, concentrated, and chromatographed on a CM-52 column sized 2.5 X 30 cm. The first two bands were eluted with a 5 mM phosphate buffer at pH 7.0, and the following six required a shallow gradient from an 8.5 to a 40 mM phosphate buffer at pH 7.0. Chromatographic separations of the covalent complexes cytfpc and Zncyt/pc are reproducible and in reasonable agreement with each other: four bands for the former and four major bands for the latter, with correspondingly similar retention times. Since the latter procedure involved larger amounts of proteins, four additional minor bands became evident. Since the presence of cytochrome c makes the complex strongly colored, separation was

304

Zhou et al.

Bloconjugate Chem., Vol. 3, No. 5, 1992

Table I. Cross-Linking of Ferricytochrome c and Cupriplastocyanin by Homobifunctional N-Hydroxysuccinimide Diesters diprotein diester protein NaCl complex(es) concn concn time concn cyt pc diester (mM) (mM) pHa (h) (M) (%I (%) 6 0 5 5 7.0 DSTb 0.40 0.050 6 0 14 12 7.0 EGSC 0.40 0.050 31 16 7.0 39 0 15 0.50 24 18 7.0 39 0.20 15 0.50 39 0 5 5 1.0 0.030 6.5 0 0 6.5 39 0.20 1.0 0.030 a A 5 mM MOPS buffer. b Disuccinimidyl tartarate. Ethylene glycol bis(succinimidy1succinate).

followed visually. Fraction collectors were unnecessary, and chromatograms were not recorded. Protein Modificationwith EDC. Cytochrome c and plastocyanin were separately incubated with EDC and chromatographed as described above for the covalent complex cyt/pc. Only the monomeric fractions from Sephadex G-75were investigated further. The cytochrome c fraction was dialyzed into an 85 mM phosphate buffer at pH 7.0 and chromatographed with it on a CM-52 column sized 2.5 X 25 cm. Three bands, with relative yields of 5, 85, and l o % , were eluted in this order. These three fractions were separately oxidized and compared with native cytochrome c for their mobility on CM-52 columns; the first fraction proved to contain the native protein. The plastocyanin derivatives were dialyzed into a 5 mM phosphate buffer at pH 7.0 and chromatographed with it on a Sephadex DEAE A-25 column sized 1.5 X 15cm. The first two bands were eluted with this buffer, and the third one required a 40 mM phosphate buffer at pH 7.0. Again, these three fractions were separately oxidized and compared with native plastocyanin in their mobility on DEAE A-25 columns; the third fraction proved to contain the native protein. Protein Cross-Linking with DST and EGS. Experimental details are summarized in Table I. Gelfiltration chromatography in all cases was done as in the EDC procedures described above. Further separations of the EGS products were tested with DEAE A-25 and CM52 columns,with various phosphate and cacodylate buffers, isocratically and with gradients. The diprotein complexes were retained by the DEAE A-25 resin. The CM-52 resin and a 1.0 mM phosphate buffer at pH 7.0 proved most effective; this chromatography gave three bands. Molecular Mass. Size-exclusionchromatography was done as in previous studies (7,22). Electrophoresis was done with a Mini Protean I1 Dual Slab Cell and supplies from Bio-Rad, Inc. Samples were denatured by the addition of SDS. A Laemmli Tris-glycine buffer and an 185% polyacrylamide gel were used. The markers had molecular mass of 6.5, 14.5, 21.5, 31.0, 45.0, 66.0, and 97.4 kDa. Protein bands were stained with Coomassie Blue. Spectroscopy. Absorption spectra at 25 f 1 OC were recorded and protein concentrations determined as in previous studies (7,lO).Each compartment of the tandem mixing cell had a pathway of 4.00 mm. The EPR spectra were recorded at 4-6 K with a Bruker ER2OOD X-band instrument. The CD and MCD spectra were recorded at ambient temperature, with a JASCO ORD/UV-5 spectropolarimeter equipped with a CD accessory and a permanent magnet. Its field strength, 600 G, was determined with K3[Fe(CN)6] as a standard (23). Molar ellipticity, [el,, in MCD experiments was corrected by making the measurements with and without the

magnetic field. Each CD and MCD spectrum was scanned at least twice, between 600 and 230 nm, at a rate of 100 nm h-l. The path lengths were 1.00 mm in MCD experiments and 1.00 mm or 1.00 cm in CD experiments. Concentrations of the samples were adjusted so that the absorbance at 410 nm was 1.06 for MCD experiments and 1.60 in a 1.00-cm cell for CD experiments. Heme exposure in native ferricytochrome c and in the covalent and electrostatic complexes cyt(III)/pc(II)was determined from the effect of ethylene glycol on the UVvis spectra (24,25),recorded in a tandem mixing cell. From the composite spectrum of the protein and of ethylene glycol, both dissolved in phosphate buffer at pH 7.0, was subtracted the spectrum of their mixture, whose final concentrations were 3.0-10.0 mM in each protein and 20 5% by weight in ethylene glycol. The buffer concentration was 85 mM in the case of the covalent complex and only 0.3 mM in the case of the electrostatic complex. The electrostatic complex was prepared by mixing the two proteins in the ratio 0.67:l.OO in order to maximize the fraction of bound cytochrome c. Both RR and SERRspectra were obtained by irradiating the samples with the 413.1 nm line of a Kr+laser (Coherent, Innova 100). The scattered light was collected in a backscattering configuration and focused onto the entrance slit of a monochromator/spectrograph (Spex, Triplemate 1877) by a pair of lenses. The spectrograph stage, containing a grating with 1800 grooves/", provided dispersion of 0.93 nm/mm and a bandpass of 10 cm-'. The spectra were accumulated and processed with an intensified diode-array detector (PARC 1420) coupled with a multichannel analyzer (PARC OMA 11). Integration time was 2 s. The spectrometer was calibrated with indene. Protein samples for RR and SERR experiments were oxidized with a small excess of dissolved &[Fe(CN)sl, dialyzed into a 5 mM phosphate buffer at pH 7.0, and concentrated; the last two operations were done by centrifugation in Amicon Centricon 3 cells. Silver sol reduced by citrate was prepared by a standard method (26),with vigorous stirring. The absorption spectrum of the sol had a maximum at 410 nm. Redox Potentials and Isoelectric Points. Differential-pulse voltammograms were obtained as in previous studies (7, 22). Spectrophotometric titrations of cytochromec were done with the K3[Fe(CN)61/K4[Fe(CN)6]couple, whose redox potential is 420 mV vs NHE; such titrations of plastocyanin were done with the [Co(trpy)zl(ClOd)3/[Co(trpy)al (ClO.& couple, whose redox potential is assumed to be 280 mV vs NHE. The two cobalt complexes were prepared by a published method (27). Oxidized and reduced forms of cytochrome c and of plastocyanin were quantitated by their absorbances at 550 and 557 nm, respectively. Isoelectric points were determined in focusing experiments with a PhastSystem from Pharmacia and with a Bio-Phoresis horizontal electrophoresis cell from Bio-Rad. Kinetics. Experiments with laser-flash photolysis were done as before (10,28,29).A phosphate buffer at pH 7.0 had ionic strength of 10 mM. Since the complex cyt(II)/ pc(I1) was in large excess over riboflavin semiquinone, simultaneous reduction of both proteins was improbable. RESULTS AND DISCUSSION

Heterogeneity of the Covalent Complexes cyt/pc. Carbodiimides soluble in water are routinely used for direct cross-linking of amino groups (Le., lysine residues) and carboxylate groups (i.e., aspartate or glutamate residues)

Protein Cross-Linking with Carbodiimide

Scheme I CYT-NH,

t

c

N-0-C-C-0-N

0 0

0

5

+ NH,-PC

BioconJugate Chem., Voi. 3, No. 5, 1992 385

-

0

n

0 0

CYT-N-C-C-N-PC H H

t

2 HO-N U

Reagent

tether-

DST

?H OH -cH-CH-

EGS

length

6h

8

9

16 A

-CH~-CH~-C-O-CH~-CHz-O-C-CH,-CH2-

according to eq 10. Application of this method to redox proteins has been critically reviewed (30). Homobifunctional N-hydroxysuccinimide diesters are used for tethering of amino groups (i.e., lysine residues) according to Scheme I. This method has also been reviewed (31).Direct cross-linking of cytochrome c and plastocyanin is one, and their cross-linking via tethers the other, aspect of the present study.

Figure 1. The a-carbon chains of cytochrome c and plastocy-

0

CYT-NH,

+

II

-0-C-PC

PC anin in the electrostatic complex. The atomic coordinates correspond to the theoretical maximum-overlap model described in ref 40.

R”=C=NHR*

0

I1

CYT-NH-C-PC

0

II

+ RHN-C-NHR’

(IO)

Cytochrome c and plastocyanin are typical proteins for they contain multiple side chains for cross-linking. Because the complex cytjpc was likely to be heterogeneous, in this study size-exclusion chromatography was followed by ion-exchangechromatography. Indeed, multiple bands were separated. Although heterogeneity was noted before in the case of direct cross-linking of cytochrome c and cytochrome c peroxidase (32, 331, to our knowledge the complex cyt/pc is the first one for which both chromatographic heterogeneity and protein configuration were examined. Electrostatic Complex cyt/pc. Because of their opposite charges, +7 and -8 at pH 7.0, ferricytochrome c and cupriplastocyanin form an electrostatic complex in solution at low ionic strength. Chemicalmodification (1820, 34, 35), electrochemical response (36), competitive inhibition (37),dependence of the cross-linking yield on ionic strength (21), NMR spectroscopy (38, 39), and computer graphics combined with electrostatic calculations (40)all clearly showed that the association involves the positively charged patch around the exposed heme edge in cytochrome c and the negatively charged patch near Tyr 83 in plastocyanin. These interaction domains are approximately defined by the lysine residues 13,25, 27, 86,and 87 in the horse cytochrome c and aspartate residues 43 and 45 and glutamate residues 44,46,60,61, and 62 in the bean plastocyanin, as shown in Figure 1. Since substitution of zinc for iron does not noticeably alter the conformation of cytochrome c (41)and its association with other proteins (15,16),electrostatic complexes cytj pc and Zncyt/pc most likely have similar configurations. But the configuration that is optimal for binding and recognition need not be optimal for reaction, as recent studies from this (7-10)and other (42)laboratories have shown. Composition of the Covalent Complexes cyt/pc. Amide cross-links probably involve the same (but not necessarily all) amino and carboxylic groups that are responsible for the electrostatic attraction. Peptide mapping proved inadequate for complete and unambiguous location of amide bonds induced by carbodiimide (30,43,

Table I1.* Properties of Ferricytochrome c,

Cupriplastocyanin, and Their Covalent Complex Formed in the Presence of the Carbodiimide EDCb property molecular mass (kDa)d EPR g values gx gYe gz gll

g1

CYt(II1) 12.5

PC(I1) 10.5

1.26 2.25 3.02 2.2 2.053

CYt(III)/PC(II) 27.0 it 1.2 1.3 2.3 3.09 2.3 2.056

reduction potential at 25 “C (mV vs NHE) Fe 256 f 2fJ 245 f 5f cu 360 f qhvi 385 f 5h Adapated from ref 7. * l-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrocholide. In 85 mM phosphate buffer at pH 7.0. Determined by size-exclusion chromatography and SDS-PAGE. e Measured at peak maximum. f Determined by differential-pulse voltammetry with 4,4’-bipyridine as mediator. 8 Reference 2. Determined by spectrophotometric titration with [Fe(CN)6I4-.i Reference 4.

441, and radiolabeling is inapplicable to this task because carbodiimide atoms are not incorporated into the crosslinks. Exact locations of the cross-links, and the static structure that it implies, are relatively unimportant because the complex cytjpc seems to be dynamical. Both the reactivity of the covalent complex, discussed in the introduction (7, I O ) , and the absence of nuclear Overhauser effects between the protein molecules in the electrostatic complex (45)point to dynamical flexibility. Fortunately, protein configuration in the covalent complex cyt/pc can be defined without pinpointing the cross-links, and other methods proved applicable. Others (21)purified the complex cyt/pc by size-exclusion chromatography and determined its composition and some properties. We corroborated their findings and added those in Tables I1 (7) and 111. The apparent molecular mass of both covalent complexes cytjpc and Zncytjpc is 27.0 f 1.2 kDa, an average of several determinations by size-exclusion chromatography and SDS polyacrylamide gel electrophoresis. This value is by ca. 17 % greater than the sum of the molecular masses of the individual proteins because molecular mass

Zhou et al.

386 Bloconjugate Chem., Vol. 3, No. 5, 1992

Table 111. Magnetic Circular Dichroism Properties of Free Ferricytochrome c and of Its Covalent Complex with Cupriplastocyanin, Formed in the Presence of the Carbodiimide EDCB protein ,A, (nm)b Ac,/H (M-l cm-l T1)c CYt(II1) 400 71 414

-77

401 414 403 417 399 414 400 414

75 -87 75 -81 61 -77 73 -81

I

cyt(III)/pc(II)derivative 1

2

3 4 0

l-Ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochlo-

ride. b f2. f10.

is only an approximate measure of the biopolymer size and because log M,is strictly proportional to elution time only for protein molecules of similar shapes. This phenomenon was semiquantitatively studied in our laboratory with diprotein complexes whose elongation varied with the size of the metal complex used for cross-linking (22, 46, 47). The visible absorption spectra confirmed the protein ratio of 1.00 f 0.08 in each of the four fractions of cyt/pc obtained by cation-exchange chromatography on CM-52 resin. Since the cross-linking reactions with the reagents DST and EGS require that the cationic lysine side chains in the two proteins face each other, and since both of these diesters hydrolyze in water, the yields of the complexes cyt/ pc are at most 15%, as Table I shows. This electrostatic repulsion is more detrimental to the shorter DST than to the longer EGS. Lower protein concentration and shorter incubation times favor the formation of the desired heterodiprotein complexes, cytlpc. Higher concentrations and longer times were avoided because they yielded the undesired homodiprotein complexes, especially cytlcyt, which cannot be fully separated from cyt/pc by sizeexclusion chromatography. The low yields of the DST complex and of the three fractions of the EGS complex obtained by cation-exchange chromatography precluded their detailed study. Raman Spectroscopy of the Covalent Complex cyt/ pc. Resonance Raman spectra (RRS) are obtained with proteins in aqueous solution. Surface-enhanced resonance Raman spectra (SERRS)are obtained with proteins adsorbed onto a roughened silver surface, but cytochrome c retains its native structure under these conditions (48, 49). Since both of these methods are noninvasive (50,51), they are well-suited to examination of possible structural perturbations of the heme and of its protein environment owing to electrostatic association and covalent cross-linking of proteins. The spectra of ferricytochrome c in Figure 2 are consistent with those reported previously (49). Peaks ~10, v2, and u3 at 1634, 1585, and 1501 cm-', respectively, are characteristic of the low-spin state. Peak v4 at 1369 cm-' is characteristic of the ferric form. The spectra of the electrostatic complex cyt(III)/pc(II) in Figure 3 differ only slightly from those of the free ferricytochrome c. The peak at 749 cm-l is absent from Figure 3A, and the relative intensities of the peaks at 397 and 410 cm-l are altered. The peak at 353 cm-l in Figure 3B has a greater relative intensity than the peak at 347 cm-l in Figure 3A. Figure 4 typifies the spectra of all four derivatives (chromatographic fractions) of the covalent complex cyt(III)/pc(II). The only apparent difference between native cytochrome

Wavenumbers (cm -l) Figure 2. Vibrational spectra of ferricytochrome c in a 5 mM phosphate buffer at pH 7.0, at room temperature. The protein concentrations are 3.3 X 10-4M for the resonance Raman spectrum (A) and 1.6 x M for the surface-enhanced resonance Raman spectrum (B).

1

t

n%

Wavenumbers (cm -I) Figure 3. Vibrational spectra of the electrostatic complex between ferricytochrome c and cupriplastocyanin in a 5 mM phosphate buffer at pH 7.0, at room temperature. The concenM for the resonance Ratrations of each protein are 1.9 X man spectrum (A) and 9.2 X lo* M for the surface-enhanced resonance Raman spectrum (B).

c and the complex is the slight decrease in the intensity of the peak at 397 cm-1 in Figure 2A, which becomes a shoulder in Figure 4A. Although the protein solutions for surface-enhanced Raman spectra are 21 times more dilute

Protein Cross-Linking wlth CarbodllmMe

Wavenumbers (cm -l) Figure 4. Vibrational spectra of the first chromatographic fraction of the covalent complex between ferricytochrome c and cupriplastocyaninin a 5 mM phosphate buffer at pH 7.0,at room M for temperature. The complex concentrations are 4.7 X M for the the resonance Raman spectrum (A) and 2.2 X surface-enhanced resonance Raman spectrum (B).

than the solutions for resonance Raman spectra, band intensities are comparable. This is an evidence of the surface effect. These Raman spectra show that ferriheme retains its six-coordinate structure and low-spin state and remains virtually unperturbed upon electrostatic association and covalent cross-linking between cytochrome c and plastocyanin. A previous resonance Raman spectroscopic study showed the same for native cytochrome f and its covalent complex with plastocyanin, formed with the reagent EDC (44). The four derivatives of the covalent complex are indistinguishable from one another and from the electrostatic complex. Number of Direct Amide Cross-Links in the Complex cyt/pc. The number of cross-links induced by EDC can perhaps be estimated from the reduction potentials. As Table I1 shows, cross-linking lowers the reduction potential of cytochrome c by ca. 10 mV and raises that of plastocyanin by ca. 25 mV (7). The following interpretation is based on the assumption that this small divergence is due mainly to neutralization of the cationic side chain@) in the former and of the anionic side chain@)in the latter. Formation of single amide bonds between one amino group of HzNCHzCHzNHz and different carboxylate groups in the anionic domain (residues 42-45 and 59-61) raises the reduction potential of spinach plastocyanin by 15-20 mV (19). Somewhat smaller increases were reported in an earlier study (52). Because the remaining amino group in ethylenediamine is protonated, this modification converts an anionic residue into a cationic one. The cross-linking with cytochrome c, however, converts an anionic residue in plastocyanin into a neutral one, so the reduction potential of plastocyanin probably increases by less than 15-20 mV per amide bond. If this protein is treated as a low-dielectric cavity with charges smeared on the surface, the expected increase is 12 mV per lost charge (53). The

Bloconjugate Chem., Vol. 3, No. 5, 1992 387

observed increase of ca. 25 mV corresponds to approximately two cross-links, on the average, in the four chromatographic fractions of the covalent complex cyt/ pc. Although the change in reduction potentials upon cross-linking may be caused by various factors in addition to charge neutralization, the preceding simple analysis should be useful. To our knowledge, no other method for estimating the number of direct amide cross-links has been proposed; this may be the first time that this difficult problem has been addressed. Other Spectroscopy of the Covalent Complex cyt/ pc. The EPR spectra are summarized in Table 11. The overlapping g, component for ferricytochrome c and the gll component for cupriplastocyanin were identified nonetheless (7). The MCD spectra, summarized in Table 111, are characteristic of low-spin ferriheme; indeed, the absorption band at 695 nm, diagnostic of axial ligation of Met 80 in cytochrome c, is evident in the spectra of all four chromatographic fractions. The electrostatic complex cyt(III)/pc(II) (a solution in a 3.0 mM phosphate buffer that is 160 pM in each protein) and all four fractions of the covalent complex cyt(III)/pc(II) gave superimposable UV-vis, CD, and MCD spectra. Although EPR, UV-vis, CD, and MCD spectra are not very sensitive to small structural changes, it is fair to conclude that electrostatic association and direct cross-linking do not significantly perturb the electronic structure of the iron and copper sites and the gross conformations of the proteins. Protein Docking in the Complex cyt/pc. The heme exposures in native ferricytochrome c, in the electrostatic complex cyt(III)/pc(II), and in the covalent complex cyt(III)/pc(II) are 24.2 f 1.3, 5.6 f 0.3, and 4.5 f 0.2%, respectively. In the first two cases, the margins of error reflect the reproducibility of the experiments; in the third case, the margin reflects the comparison among the four chromatographic fractions of the covalent complex. These findings confirm the previous findings by others (see above) that plastocyanin covers most of the exposed heme edge in cytochrome c. Moreover, the four fractions of the covalent complex seem to be structurally similar to one another and to the electrostatic complex. When the acidic patch is blocked by chemical modification, plastocyanin fails to form the covalent complex with cytochrome c in the presence of EDC. All the fractions separated on the CM-52 column consisted of monomeric proteins (modified by the carbodiimide, as explained in the next subsection). Evidently it is the acidic patch that covers the heme edge in the covalent complex. The interaction domains in the cross-linked proteins are most likely the same as those determined for the electrostatically associated proteins, namely lysine residues 13,25,27,86, and 87 in the horse cytochrome c and aspartate residues 43 and 45 and glutamate residues 44,46,60,61,and 62 in the bean plastocyanin. The intracomplex reaction in eq 1 is undetectably slow (less than 0.2 s-l) in all four fractions of the covalent complex (7,10). The lifetime of the triplet state is sensitive to protein docking, yet it is the same, 5.2 f 0.2 ms, in all eight fractions of the covalent complex 3Zncyt/pc(I) (9). The rate constant kffor the intracomplex reaction (eq 2) is also the same, (2.2 f 0.5) X lo4s-l, in all eight fractions of the covalent complex 3Zncyt/pc(II) (9).This uniform unreactivity, uniform photophysical properties, and uniform reactivity are additional evidence for structural uniformity of the fractions into which the covalent complex cyt/pc separates on the CM-52 resin. Chromatographic Heterogeneity of the Covalent Complex cyt/pc. Since the diprotein complexes cyt(III)/

Zhou et ai.

388 Bloconjugate Chem., Vol. 3, No. 5, 1992

pc(I1) and Zncyt/pc(II) are eluted from the cation exchanger CM-52 by dilute phosphate buffers, their overall charges must be relatively low. Indeed, the opposite charges of the individual proteins nearly neutralize each other upon association. Mobility of individual chromatographic fractions is characteristic of differences in total charge and in the distribution of charge (47). Besides efficiently and noninvasively cross-linking proteins, carbodiimide also modifies side chains (54, 55). Indeed, reaction of cytochrome c with EDC yields derivatives in which carboxylic groups, which are anions in neutral solution, have been converted into neutral N-acylurea groups according to eq 11 (56). We confirmed these

I

0

I1

PROTEIN-C-OH

I

+

R”=C=NHR+

1 4

0

II

II

1

2

4

6

I 8

SECONDS

0

PROTEIN-C-NR‘-C-NHR

I

I

(11)

findings and also found similar conversions with plastocyanin. These derivatives of the individual proteins resembled the fractions of the covalent diprotein complex in their relative mobility on the CM-52 column. The attempt to cross-link cytochrome c to plastocyanin whose acidic patch had previously been modified by ethylenediamine yielded only the N-acylurea derivatives of the separate proteins. Isoelectric points confirm that chromatographic fractions of the covalent complex differ from one another in charge. The values for the complex cytlpc are 6.2,6.8,7.1, and 8.2; those for the complex Zncytlpc are 6.2, 7.1, 8.0, 8.2,8.5,9.0,9.6, and >9.6. Both of these series parallel the trends in mobility on the cation exchanger CM-52. These chromatographic fractions are derivatives with different numbers and locations of N-acylurea groups. This conclusion is supported by our detailed comparison of individual derivatives of the covalent complex Zncyt/pc(I) in their electron-transfer reactions according to eq 7 (12). Since peptide mapping is inapplicable to cross-linked complexes,these groups cannot be conclusivelyidentified. Mass spectrometry with electrospray ionization (57,58) may in the future become routinely applicable to protein complexes, but even this method would permit only quantitation, not location, of N-acylurea groups. Their identification in the derivatives of cytochrome c and of plastocyanin would be of little value because functional groups that are accessible in separate proteins may be inaccessible in the diprotein complex. Indeed, the change in the UV-vis spectrum that was attributed to modification of a propionate group in cytochrome c (56)is absent in the spectra of the cytlpc derivatives. This propionate group, which is attached to heme and somewhat exposed to the protein exterior in native cytochrome c, becomes shielded when plastocyanin covers the heme edge. The rate constants for the reactions in eqs 1and 2 are the same for all the derivatives. Since the lifetime of the triplet excited state and the intracomplex electron-transfer reactions do not depend on the number and location of N-acylurea groups on the surface, these groups were not examined further. Electron-Transfer Reactions of Tethered Complexes cytfpc. The rate constant at the ionic strength of 10 mM for the external reduction by riboflavin semiquinone is 5.4 X lo7 M-1 s-l in the case of native ferricytochrome c (59) and 1.1 X lo7 M-’ s-1 in the case of the diprotein complex cyt(III)/pc(II) tethered by EGS (eq 12).

Figure 5. Reaction of riboflavin semiquinone with the covalent complex cyt(III)/pc(II) in which the protein molecules are tethered with EGS. Conditions: 10 MM cyt(III)/pc(II) in a phosphate buffer at pH 7.0, ionic strength 10 mM; absorbance at 550 nm.

Since riboflavin semiquinone reacts near the exposed heme edge (59), the inhibition of its attack at the diprotein complex indicates that the tether is attached to this area of cytochrome c surface, which is especially rich in lysine residues. Figure 5 shows the initial curvature, corresponding to the external reaction (eq 12) and a horizontal plot thereafter, corresponding to the lack of the internal (intracomplex) reaction (eq 1) at this low concentration of the diprotein complex. At higher concentrations the intercomplex reaction (eq 8)becomes evident. The complex cyt(III)/pc(II) tethered with DST gave similar absorbance traces. The complexes cytfpc with long tethers behave exactly like the complex cytfpc with direct amide cross-links (7, IO). None of them undergoes the thermodynamically favorable internal reaction (eq 1). Although tethers allow some mobility of the protein molecules, the complexes apparently cannot adopt the reactive configuration (9,421. Only the electrostatic complex cyt/pc, in which surface diffusion of the interacting protein molecules is unrestricted, can achievethe optimal configuration and undergo the internal reaction at a relatively fast rate (7, IO). CONCLUSION Direct cross-linking of proteins with carbodiimides produces multiple derivatives of the protein complexes, which need to be separated, purified, and examined. When the proteins form only one major electrostatic complex in solution or when multiple electrostatic complexes have similar configurations, the covalent complex is likely to be structurally homogeneous or heterogeneous beyond detection; this is the case with cytochrome c and plastocyanin. But when the proteins form different electrostatic complexes in appreciable amounts, structural heterogeneity of the covalent complex may be sufficient to allow separation of isomers; this may be expected of proteins that have on their surfaces multiple complementary patches of opposite charge. In either case, the covalent complex is likely to be heterogeneous in terms of net charge and of charge distribution on the surface. This heterogeneity may affect the interactions of the complex with other biomolecules and membranes. ACKNOWLEDGMENT This work was supported by NSF through apresidential Young Investigator Award to N.M.K. (Grant CHE8858387). We thank Dr. Venkatesh M. Shanbhag for dis-

Protein Cross-Linking with Carbodiimide

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