Segal, S y k e s
/ Blue Copper Protein Electron-Transfer Reactions
4585
Kinetic Studies on 1:1 Electron-Transfer Reactions Involving Blue Copper Proteins. 1. Evidence for an Unreactive Form of the Reduced Protein (pH < 5 ) and for Protein-Complex Association in Reactions of Parsley (and Spinach) Plastocyanin Michael G. Segal and A. Geoffrey Sykes* Contribution f r o m the Department of Inorganic and Structural Chemistry, The Unicersity, Leeds LS2 9JT. England. Receiced October 21, 1977
Abstract: Reactions involving the 1:l oxidation of reduced parsley plastocyanin, PCu(I), with C0(phen)3~+,C0(bpy)3~+.and Fe(CN)63-, and reduction of the blue PCu(l1) with Fe(CN),j4-, R U ( N H ~ ) ~ P Yand * + , R u ( " ~ ) ~ ~ + , have been investigated, I = 0.10 f 0.01 M (NaCI), by the stopped-flow method. Whereas overall rate constants for reactions of PCu(I1) with Fe(CN)64- show little variation (ca. 30% increase) on decreasing the pH from 7 to 5, there is a dramatic decrease for reactions of PCu(I) with all three oxidants corresponding to rapid (reversible) formation of a redox-inactive protonated form of the protein with acid dissociation constants, pK,, in the range 5.7-6.1. Protonation at or near to the Cu(1) site seems likely. Overall activation parameters, pH >7, were determined for all but the R u ( " ~ ) ~ ~ +reaction, and in two cases it was possible to extend the range of complex concentrations sufficiently for (kinetic) detection of protein-complex association ( K ) prior to electron transfer ( k c t ) .These were the reactions: PCu(1) C0(phen)3~+,where at 25 "C, K = 167 M-I, A H o = I O kcal mol-', A S o = 45 cal K-' mol-', k,, = 17.9 s-l, AH,,* = 4.3 kcal mol-', AS,,* = -39 cal K-' mol-!; and Fe(CN)64PCu(ll). K = I10 M-I, A H o = -5.1 kcal mol-', A S o = -7.8 cal K-' mol-', k,, = 170 s-l, AH,,* = 11.4 kcal mol-', AS,,* = -9.7 cal K-! mol-'. Comparisons with previous data for the temperature-jump equilibration study of azurin ACu(l)/ACu(ll) with Fe(CN)64-/Fe(CN)63- are made. Possible limitations imposed by consideration of overall kinetic parameters without reference to K and k,, are to be stressed. From ionic strength dependences and values of A H o and A S o as above the protein behaves as a negatively charged species, but it is not clear whether this corresponds to the charge a t the binding site. A similar dependence on pH and evidence for association were obtained in the C0(phen)3~+oxidation of spinach plastocyanin.
+
+
for 24 h at ca. 0 OC. To obtain the reduced form, PCu(I), a few crystals Plastocyanin is a blue copper (type 1 ) protein which is of sodium dithionite (G.P.R. grade, B.D.H.),representing an excess found in plant chloroplasts and other photosynthetic organof this reductant, were added before dialysis. Reduction is known to isms.' It is an essential component in the photosynthetic elecbe rapid.8 The reduced form has the same CV spectrum but no visible tron-transport chain making use of the blue Cu(I1) and colabsorption. orless Cu(1) states.2While earlier papers have indicated that Spinach plastocyanin was extracted from spinach leaves by the there are 2 Cu atoms per protein it has now been demonliterature method.'! After removal of ferredoxin as described, the 0.2 ~ t r a t e dthat ~ , ~there is only a single C u atom per unit of moM CI- eluate of the first DEAE-cellulose (Whatman Ltd.) column lecular weight ca. 10 500. The Cu is bound in a distorted tetwas diluted 2X, loaded onto a column of DE23 cellulose (7 cm long, 5 cm diameter), and washed with 0.05 M sodium phosphate buffer rahedral ligand en~ironment,~ which is not accessible to solvent (pH 6.9) and the plastocyanin PCu(I) was eluted with a 0.1 M solution H20.6 Plastocyanins from different plant sources show only minor differences in amino acid composition and s e q ~ e n c i n g . ~ . ~of the same buffer. The protein was purified in the same way as for parsley on further columns of DEAE-cellulose. The spectrum is very An isoelectric point is observed a t pH ca. 4.2,' and at higher similar to that of parsley, Figure 1, peak ratio a278/A597 = 1.69. pHs the protein becomes appreciably negatively charged. Spectra are compared with those of other blue Cu proteins in Table Standard reduction potentials have been determined and are I. ca. 360 mV.899 Complexes. Tris( l,lO-phenanthroline)cobalt(III) was prepared As a means of understanding more fully features of the as the perchlorate salt, [C0(phen)~](C10~)3.2HzO,~~ which was electron-transfer process involving such metalloproteins, repercipitated out of solution by addition of saturated NaC104, and actions of parsley and (to a lesser extent) spinach plastocyanin recrystallized from HzO with the addition of a few drops of 5 M HC104. Peak positions [A, nm (c, M-I cm-I)] were at 330 (4660), have been investigated, and are reported herein. Comparisons 350 (3620), and 450 (100) in good agreement with literature values. with studies involving the blue copper protein azurin from The chloride salt, [C0(phen)3]C1~.7HzO,'~ was also used when the bacterial sources are made. The abbreviations PCu(I)/PCu(II) perchlorate salt was not sufficiently soluble. Replacement of CI- by and ACu(I)/ACu(II) are used to indicate different oxidation C104- was shown to have no effect on rate constants (see e g , Table states of plastocyanin and azurin, respectively. I l l of the microfilm edition; see paragraph at end of paper). Tris(2,2'-bipyridine)cobalt(III), [Co(bpy)3](C104)3-3HzO, was Experimental Section
Proteins. Plastocyanin was isolated from parsley leaves by the method of Plesnitar and Bendall.lo After preparation the parsley leaves were stored at ca. -15 "C for a minimum time (generally overnight) before use. The final stage in the procedure described, involving concentration of the protein, was omitted. The yield of PCu(1I) M solution from 2.5 kg of cut leaves. was 20-40 mL of (4-6) X This gave an absorbance ( A ) ratio at peak positions A278/A597 of 1.7 f 0.1, in good agreement with previous determinations.8 The spectrum based on c = 4.5 X IO3 M-I cm-l at 597 nm7,8is as shown in Figure I . Concentrations were determined from the absorbance at 597 nm. N o significant ( f 5 % ) variation in kinetic parameters was observed using samples from different batches. Before use the protein solution was dialyzed (Sigma 21-mm dialysis sacks) against the required buffer
0002-7863/78/1500-4585$01.00/0
prepared,I4 and gave a spectrum with peak positions [ A , nm (c, M-I cm-I)] at 306 (34 IOO), 317 (30 700), and 450 (68.4) in agreement with previous values. Potassium hexacyanoiron(III), K3Fe(CN)6 (B.D.H, AnalaR), peakat 300 (1600) and 420 (1010). and potassium hexacyanoiron(II), K4[ Fe(CN)6].3HzO (Hopkin and Williams, AnalaR), peak 330 (330). were used as supplied. Solutions of Fe(CN)64- were used soon after preparation, i.e. within 30 min, or alternatively were stored under Nz. When this precaution was not observed significant air oxidation occurred, and there was an increase in experimentally determined rate constant due to formation of Fe(CN)6)-. Hexaammineruthenium(1 11) chloride, [Ru("3)6]Cl3, was obtained from Johnson and Matthey and purified by the literature method.I5 Aqueous solutions (1-5) X M were reduced (ca. 75% conversion at neutral pH) on an amalgamated zinc shot column (20
0 1978 American Chemical Society
Journal of the American Chemical Society
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Table I. A Comparison of Visible Spectra [ A (nm), t (M-I cm-I)] of Blue Copper Proteins, Oxidized From Cu(II), in Aqueous Solution, D H -7 A
Protein
Source
Plastocyanina Plastocyaninb Azurin Stellacyaninb
Parsley Spinach Pseudomonas A Rhus vernicifera
(€1
(6)
460 (600) 460 (590) 459 (464) 448 (554)
(e)
770 ( 1 500) 770 (1650) 781 (521) 845 (700)
597 (4500) 597 (4900) 625 (5700) 604 (3820)
a This work; see also ref 7 and 8. R. Malkin and B. G . Malmstrom, Ado. Enzymol.. 33, 177 (1970).The protein unit is assumed to contain I Cu (mol wt I O 500). Azurin c values as in ref 32.
+
Fe(CN)64- PCu(l1) (present study) is in good agreement with a value of ca. 7 obtained from redox potentials, thus supporting a oneelectron reaction. McArdle et a1.2' have previously studied the oxidation of PCu(l) with C0(phen)3~+and analyzed their data in terms of a 1:1 reaction. Spectrophotometric titration of PCu(l) with Co(phen)13+ indicated a I : l reaction with an equilibrium constant for PCu(1) C0(phen)~3+e PCu(lI) C0(phen)3~+of 1.6 in close agreement with the electrode potentials quoted above, pH 7.0 (phosphate), I = 0.10 M (NaCI). Kinetic Studies. Ionic strengths ( I ) were adjusted to 0. I O M using NaC1. For reactions in M sodium phosphate at pH 7.0we have followed Wood* in using 0.09 M NaCI. With the phosphate contribution this yields a total ionic strength of ca. 0.108 M. Reactions were monitored using a Durrum stopped-flow spectrophotometer. Traces were photographed from a Tektronix storage oscilloscope, where at least two traces were analyzed for each run (agreement &5%). We attempted to study the equilibration of Fe(CN)64- with PCu(ll) by temperature-jump techniques, but encountered stability problems with I 0 L L the protein, which was decolorized after a series ofjumps. Instead it 500 700 h(nm) was found that the reactions of PCu(l) with Fe(CN)63-, and Fe(CN)64- with PCu(ll), could be studied independently by the Figure 1. UV-visible spectrum of parsley plastocyanin PCu(II) in aqueous stopped-flow method by having reactants Fe(CN)63- (>tenfold) and solution, pH 7.The reduced form PCu(I) has the same spectrum, 30-fold), respectively, in excess. In all experiments the nm. formation or decay of the blue Cu protein absorbance at the 597-nm was monitored with time. Plots of log AA against time were linear for at least 3 half-lives. First-order rate constants were obtained from slopes (X2.303).Air-free conditions (Argas) were necessary for the cm long, 2 cm diameter) under argon gas, immediately prior to use. Ru(NH3)5py2+ and R u ( N H ~ ) ~studies. ~ + The latter reduction is very Concentrations were determined by reacting with F-superoxorapid and had to be studied under second-order conditions with bis[pentaamminecobalt(lll)], when there is a rapid 1:l reaction, and [Ru(NH3)6'+] = (1-6)X M a n d [PCu(ll)] = (7-10)X measuring the absorbance change a t 670 nm ( e 890 M-l cm-l). M to obtain meaningful traces. Under these conditions rate constants Crystalline pyridinopentaammineruthenium(l1) perchlorate, obtained were lacking in precision and only an approximate rate [Ru(NH3)5py](C104)2, was prepared by the literature method,I6 constant is indicated. starting from [Ru("&]C13. Peak positions A, nm ( e , M-'cm-l)] Treatment of Data. A nonlinear least-squares programZZand subwere at 244 (4.8X IO3) and 407 (7.24X IO3). routines based on this were used. Weighting factors were in all cases Buffers. Solutions were prepared from mono- and disodium hyequivalent to 1 / y 2 . For computation of data in connection with eq 1, drogen phosphates, hydrochloric acid, sodium acetate, and acetic acid ko, k H , and K H were independent variables; with eq 7,AH', AS' (for (all AnalaR, B.D.H.), sodium cacodylate, Na[(CH3)2As02] (LabK ) , AH,,*, and AS,,* (for ke,) were independent variables. oratory Reagent, B.D.H.), and tris(hydroxymethy1)aminomethane (Trizma) here referred to as Tris (Sigma Chemicals). For experiments Results M sodium cacodyin which the pH variation was investigated, Parsley plastocyanin was used throughout except for the late, pH adjusted to 5.0-7.5by addition of HCI, was used. Tris was M HCI to give solutions pH 7.0-9.0, and 5 X added to 2 X oxidation of PCu(1) with Co(phen)j3+ when the reaction (25 M sodium acetate with HCI was used to obtain a pH of 4.3.Rate "C) with spinach plastocyanin was also studied. constants in Tris were found to be 20% higher than those in cacodylate Oxidation of PCu(1) with C0(phen)3~+.Second-order rate for the region where the pH ranges overlapped (see, e.g., Table IXZ3). constants, k , for the reactions of both parsley and spinach T o investigate complex concentration dependencies, and for the deplastocyanin with C0(phen)3~+(7.5. Rate constants, k , a t p H 7 (Table VII) give a n estimate for ko. A least-squares fit of data at pH 7, 10.0-29.6 "C, gives k(25 "C) = 322 M-I s-I, AH* = 9.7 f 4 kcal mol-', AS* = -14.6 f 1.5 cal K-I mol-'. On increasing the range of [C0(bpy)3~+]to 4.0 X M no evidence for association as in eq 5-6 was obtained. Table VI11 gives the variation of k with ionic strength.23 Oxidation of PCu(1) with Fe(CN)63-. Second-order rate constants, k (Table IXz3) give a dependence on pH, Figure 4, similar to that for C0(phen)3~+.A fit of data to the same type of reaction sequence yields K H = (5.2 f 0.2) X l o 5 M-I, ko = (7.8 f 0.2) X lo4 M-] s-l. Theconcentration of Fe(CN)63was increased to the limit possible for stopped-flow studies without being able to detect a less than first-order dependence on oxidant [Fe(CN)63-]. It was not possible therefore to evaluate K and ket. From the temperature dependence 10.5-33.4 "C, Table X, k(25 "C) = (9.4 f 0.4) X lo4 M-I s-l, AH* = -3.4 f 0.1 kcal mol-', and AS* = -47 f 0.3 cal K-I mol-'. Thevariation of k with I was investigated (Table ~123).
Reduction of PCu(I1)with Fe(CN)64-. Only a small variation in rate constant k with pH was observed, Figure 4 (Table
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Journal of the American Chemical Society
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.,0-'[FdC N:64-]-'( M -')
PH
Figure 4. The variation of rate constants (25 "C) for the reactions of parsley plastocyanin PCu(1) F ~ ( C N ) G(~0-)and Fe(CN)64- PCu(I1) (0)with pH, I = 0.10 M (NaC1).
+
+
Figure 5. The linear dependence of reciprocal first-order rate constants for the Fe(CN),j4- reduction of parsley PCu(I1) on [Fe(CNh4-] at pH 7.0, I = 0.108 M (NaC1).
Table X. Second-Order Rate Constants for the Oxidation of PCu(1) Parsley Plastocyanin (10-5 M) with Fe(CN)63- at pH 7.0a and pH 5.0b
Table VII. Second-Order Rate Constants for the Oxidation of PCu(1) Parsley Plastocyanin M) with C0(bpy)3~+at pH 7.0a and pH 5.0b
Temp, OC 29.6 25.0
7.0
1.50 0.20 0.60
1 .oo 1S O 2.00 3.00 4.00 0.60
5.0
1.oo 1 2.00 1S O 1S O 1S O
.so
20.4 15.2 10.0
7.0 7.0 7.0
33.4 29.5 25.0
412 313 297 310 (2) 326 317 (2) 342 354 45.4 52.0 52.3 52.0 26 1 173 129
18.6 10.5 25.0
PH
104[Fe(CN)63-], M
7 .O 7.0 7.0 7.0 7.0 7.0 7.0 7.0 5.0 5.0 5 .O 5.0
1.28 1.28 1.28 2.54 4.35 5.55 1.28 1.28 1 .oo 2.00 4.00 7.00
10-4k, M-I s-I 7.58 8.20 8.75 9.24 9.85 9.63 9.61 11.1
2.29( 2.19 2.09 2.10
a Buffer 10-2 M sodium phosphate; I = 0.108 M (NaCI). Buffer IOV2 M sodium cacodylate HCI; I = 0.10 M (NaC1); PCu(1) stored V. a t pH 5.0. c [PCu(I)] = 5 X
+
a M sodium phosphate, 1 = 0.108 M (NaCI). b 10-2 M sodium cacodylate HCI, I = 0.10 M (1VaCI).
+
XI123). On extending the range of [ F e ( c N h 4 - ] to (0.3-2.0) X loh3 M, first-order rate constants, k&sd (Table XII123),gave a nonlinear dependence on [Fe(CN)64-]. Plots of kobsd-l against [Fe(CN)64-]-1, Figure 5, are linear, consistent with the reaction sequence of eq 8 and 9, which is similar to eq 5 and 6: K
Fe(CN)64- t PCu(I1) + Fe(CN)64-, PCu(I1)
(8)
k't Fe(CN)63- + P c u ( I )
(9)
Fe(CN)64-, P c u ( I I )
From a least-squares fit of data, 6.8-29.4 "C, K(25 "C) = 110 f 30 M-I, AH" = -5.1 f 2.6 kcal mol-', ASo= -7.8 f 9.0 cal K-' mol-', ket(2.5 "C) = 170 f 40 s - I , AHet* = 11.4 f 2.3 kcal mol-], ASet* = -9.7 f 8.0 cal K-I mol-'. The ionic strength dependence was also investigated (Table XIV23). Reduction of PCu(I1) with Ru(NH&py2+ and Ru(NH&~+. Of prime interest was whether a nonlinear dependence o n reductant could be detected. Unfortunately both reactions were too rapid for a sufficiently extensive range of reductant concentrations to be investigated. Overall activation parameters were obtained from studies on the reaction of Ru(NH3)5py2+
(7.1-25 "C),TableXV,Thesearek(25 "C) = (1.1 f 0 . 1 ) X lo6 M-I s-l, AH* = 9.7 f 0.8 kcal mol-], AS* = 1.6 f 2.9 cal K-I mol-'. Rate constants for the Ru(NH3)6*+ reduction of PCu(I1) a t 5 "C were ca. 2.4 X 106 M-I s-I in Tris buffer (pH 7.0).
Discussion Gray and coworkers25 have previously investigated the effect of p H on the Fe(edta)2- reduction of bean PCu(I1) and found there to be a (relatively) small effect in the range pH 5-8. For the oxidation of PCu(1) no similar investigation of pH has been reported. A similar dependence to that for PCu(I1) would be consistent with the report by Katoh et ala9that the reduction potential for spinach PCu(II)/PCu(I) is invariant until p H 7, the behavior can otherwise be satisfactorily accounted for in terms of a simple protonation as in eq 2 with the protonated form redox inactive. Rate constants for the Fe(CN)63- oxidation of PCu(1) decrease from 7.9 X lo4 M-I s-’ at p H >7 to zero at pH
&‘loo+
&
,
b
I 3
5
5
0
9
1
PH
Figure 6 . The variation of reduction potential for the PCu(lI)/PCu(I) couple with pH. Points ( 0 )are as derived from kinetic data for the respective reactions with Fe(CN)& and F ~ ( C N ) G ~The - . line drawn deviates from these points at low pH assuming t h a t the PCu(I1) Fe(CN)63- reaction is independent of pH and t h a t the rate constant pH 7 applies. The points (0)are taken from Katoh et al. (ref 9).
+
for the above effect. However, an alternative explanation which we find attractive is that addition of a proton may bring about dissociation of a ligand from the metal. Crystallographic evidence for coordination of the Cu to one cysteinyl sulfur, two histidine nitrogens, and one methionine at a distorted tetrahedral site 6 8, from the surface of the protein has been obtained.5 In the absence of a crystallographic study the same structure is assumed to apply for PCu(I), which is in keeping with the extremely efficient interconversion of PCu(1) and PCu(I1) a t p H 3 7 involving minimal reorganizational requirements. A distorted tetrahedral geometry is believed to be acceptable to both oxidation states.*’ From its coordination chemistry Cu(1) exhibits 2,3, and 4 (tetrahedral) coordination. Coordination numbers of 328 and 429 are only generally acceptable, however, in the presence of II-acceptor ligands in view of the 3d1° configuration. Dissociation of the methionine ligand would not involve a proton since the sulfur atom remains unprotonated. Retention of the cysteine ligand as a stabilizing influence seems likely. From N M R studies protonation of the two histidines of PCu(1) has been reported with pK, values of 4.9 and
