S y k e s et ai.
/
2291
Oxidation of Parsley Plastocyanin
Kinetic Studies on 1: 1 Electron-Transfer Reactions Involving Blue Copper Proteins. 2. Protonation Effects and Different Binding Sites in the Oxidation of Parsley Plastocyanin with Co( 4,7-DPS~hen)3~-, Fe( CN)63-, and Co( ~ h e n ) 3 ~ + ? A. Graham Lappin, Michael G . Segal, David C. Weatherburn, and A. Geoffrey Sykes* Contribution f r o m the Department of Inorganic and Structural Chemistry, The Uniuersity, Leeds L S 2 9JT, England. Received August 30. I978
Abstract: Strong protein-complex association ( K ) prior to electron transfer ( k , , ) is observed in the oxidation of parsley plastocyanin, PCu', with the 4,7-di(phenyl-4'-sulfonate)-1 , I 0-phenanthroline complex, C0(4,7-DPSphen)3~-.At 25 "C, I = 0.10 M (NaCI), K = 4600 M-I, AH" = -4.2 kcal mol-', ASo = 2.7 cal K-' mol-', k,, = 0.041 s-I, AH*,, = 13.2 kcal mol-', AS*,, = -20.6 cal K-' mol-'. Rate constants are independent of pH in the range 5.2-7.5 investigated. This contrasts with the strong p H dependence observed with C0(phen)3~+and Fe(CN)63- as oxidants (pK, 6). Addition of redox-inactive Cr(phe~1)3~+ (which forms a 1:l adduct with the protein) blocks reaction with Co(phen)j3+, consistent with a single binding site being utilized by this oxidant. Rate constants for the Fe(CN)63- oxidation of PCu' a t pH 7.0 are unaffected by the presence of C o ( 4 . 7 - D P S ~ h e n ) ~which ~ - is l : 1 associated (up to 70%) with the protein. It is concluded that C0(4,7-DPSphen)3~-and Fe(CN)63- use different binding sites on the protein. Moreover, for the reaction of C ~ ( p h e n ) ~with ~ + PCu' it has been shown that on decreasing the pH from 7.5 to 5.5 K decreases significantly, whereas k,, increases slightly. For this reaction the de0. Such an effect suggests that H+ modifies the bindcrease in overall rate constants to zero at pH ca. 5 is therefore due to K ing site on the PCu'. A similar change is not observed with PCu". These results indicate that different binding sites have different reduction potentials.
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The plastocyanins are copper proteins (mol wt ca. 10 500) containing type 1 copper,',? which lie partially exposed on the surface of the thylakoid membrane,3 where they are involved in electron transport from photosystem I1 to photosystem I.4 They contain a single copper which utilizes oxidation states 1 and 11. The molecular structure of poplar plastocyanin PCu" has recently been determir~ed,~ and the Cu(I1) shown to be bound by two histidines, one cysteine, and one methionine in a distorted tetrahedral arrangement. Similar structural features are expected to be retained in other PCuI1 species, and also in the reduced protein PCu', the structure of which is at present being i n v e ~ t i g a t e d . ~ The work of Gray and colleague^^.^ has helped establish the area of investigation of redox reactions of blue copper proteins using inorganic complexes as redox partners. Results with parsley and spinach plastocyanin previously reported from this laboratory8 have provided evidence consistent with a mechanism involving association of the protein and metal complex prior to electron transfer. PCu'
K + oxid + PCu', oxid
(1
1
kei
PCu', oxid +products
(2)
A similar mechanism is involved in the reduction of PCu". Factors influencing protein-complex association, protonation effects, and the nature of binding sites are further investigated in this paper.
Experimental Section Protein. Plastocyanin was isolated from parsley leaves by the method of PlesniEar and BendaL9 It was purified and stored (ca. 5 X M ) as previously described.8 The oxidized form, PCu1I,gave an absorbance peak ratio A278/A597 = 1.7 0.1. T o obtain the reduced protein, PCu', a few crystals of sodium dithionite (GPR grade, BDH), representing an excess of reductant, were added before dialysis. Protein solutions were dialyzed (21-mm diameter sacks, Sigma) against the appropriate buffer for 30 h at 0 "C. t No reprints available
0002-7863/79/1501-2297$01 .OO/O
Complexes. The sodium salt of tris[4,7-di(phenyl-4'-sulfonate)1,IO-phenanthroline]cobalt(lll),C0(4,7-DPSphen)3~-,was prepared
4,7 DPSphen a s described elsewhere and purified by repeated precipitation from aqueous ethanol.'Osi' The dried, purified form had an absorbance maximum at 293 nm, e 1.2 X I O5 M-' cm-I, based on the formula Na3[Co(4,7-DPSphen)!], in agreement with the literature va1ue.I' Tris( 1 ,IO-phenanthroline)cobalt(lI I ) chloride and perchlorate salts were prepared as previously described.* Tris( 1 ,IO-phenanthro1ine)chromium(ll1) perchlorate, [Cr(phen)3](C104)3, was prepared by a method based on that of Lee et al.I2 A solution of I,l0-phenanthroline monohydrate (0.60 g) in absolute ethanol (ca. 12 mL) was deoxygenated with N2 using air-free techniques. Chromium(l1) (ca. 0.8 M) in HC104 (ca. 0.8 M) was prepared by reduction of chromiu m ( l l l ) with amalgamated zinc shot under N2, and 1.2 mL injected into the solution of ligand with stirring. A solution of 1 2 (ca. 0.1 5 g) in ethanol (7 mL) was added, the mixture extracted with boiling water (ca. 120 mL), and NaC104 (2 g) added (in air). After the resulting solution was cooled the product was filtered off, washed with a little cold water and ethanol, and recrystallized from water. The pure product was washed with ethanol and dried in vacuo over P205. Analyses for C , H , and N were satisfactory. The UV-visible spectrum gave h, nm (e, M-] cm-l) at 430 sh (642), 320 sh (1.32 X lo4), and 266 peak (6.37 X lo4),in good agreement with the literature spectrum.13 Potassium hexacyanoiron(1 [ I ) , K3Fe(CN)6 (BDH Analar), peaks h, nm (e, M-' cm-l) a t 300 (1600) and 420 ( l o l o ) , was used. Buffers. Phosphate and cacodylate buffers were as described previously.8 Acetate buffers were prepared from sodium acetate (Analar, BDH) and hydrochloric acid. Collidine (2,4,6-trimethylpyridine, Laboratory Reagent, BDH) was also used with hydrochloric acid. The M with phosphate, buffer concentration after mixing was 1 X cacodylate, and acetate. With collidine, solutions were adjusted to keep
0 1979 American Chemical Society
2298
Journal ofthe American Chemical Society
30t
5
0
10
io*tc~(m)i(MI Figure 1. The variation of first-order rate constants, k o b s d , with Co(lll) concentration for the C0(4,7-DPSphen)3~-oxidation of reduced parsley plastocyanin, PCuI, at pH 7.50 (IO-* M phosphate), I = 0.10 M (NaCI).
the concentration of the protonated form of the buffer constant at I X IO-2 M. Normally 1 X M buffer was present in both reactant solutions and the p H was adjusted to the required value at least 30 min before mixing. For a series of experiments in which the pH was varied, 2 X IO-* M buffer a t the desired pH was present in the oxidant solution, and the protein solution contained l X M buffer at pH ca. 7 . The pH of solutions after mixing was measured using a Radiometer ( P H M 4d) instrument with a combined electrode type G W 2322C.
Kinetic Studies. Ionic strengths ( I ) were adjusted to 0. I O M using NaCI. Reactions were monitored at the visible absorption maximum for PCu" at 597 nm, 6 4.5 X IO3 M-' cm-',8usinga Durrum-Gibson stopped-flow spectrophotometer. For the slower reactions the added precaution of closing all taps to the absorption cell after mixing substantially prevented any diffusion. Traces were photographed from a Tektronix (RM564) storage oscilloscope and at least three traces were analyzed for each run. A large excess (>tenfold) of oxidant was used in all runs. Plots of log ( A , - A , ) against time were generally linear for at least 3 half-lives and first-order rate constants, kobsd, were obtained from the slopes (X2.303). For some C0(4,7-DPSphen)3~runs the Guggenheim method was used.I4 Treatment of Data. A nonlinear least-squares programI5 and subroutines were used. Weighting factors were 1 / y for the slower (less accurate on stopped-flow time scale) reactions and l / y 2 in all other cases.
Results Oxidation of PCu(1)with C0(4,7-DPSphen)3~-.First-order rate constants kobsd (pH 7.50), Table I,I6 are dependent on [C0(4,7-DPSphen)3~-]as shown in Figure 1. Plots of (kobsd)-' against [C0(4,7;DPSphen)3~-]-~are linear with positive intercepts, consistent with a mechanism as in ( 1 ) and (2). A nonlinear least-squares fit to the derived rate law Kket 11)I (3) 1 K[Co(III)] gave K(25 "C) = 4600 M-I, AH" = -4.2 f 1.8 kcal mol-', AS" = 2.7 f 5.9 cal K-I mol-', and k,, (25 "C) = 0.041 s-l, AH*,, = 13.2 f 0.8 kcal mol-', AS*,, = -20.6 f 2.5 cal K-I mol-'. Rate constants, kobsd, show no dependence on [H+] over the p H range 5.2-7.5, Figure 2, and no effect on changing the buffer from cacodylate to phosphate. Oxidation of PCu(1)with C0(phen)3~+. Previous studies have indicated that in cacodylate buffer there is a strong p H dependence of the reaction between PCul and C0(phen)3~+, kobsd
=
+
/ V
101:9
/ April 25, 1979
A
1""
Figure 2. The dependence of rate constants on pH for the oxidation of reduced parsley plastocyanin, PCu', with cobalt(ll1) complexes at 25 OC. Left-hand ordinate: second-order rate constants for the reaction with C0(phen)3~+in cacodylate ( O ) , acetate (e),collidine (A),and phosphate (V).Right-hand ordinate: first-order rate constants for the reaction with C0(4,7-DPSphen),~-( 2 . 5 X 10-4 M) in phosphate (0)and cacodylate (m) buffers.
Figure 2. The absence of a comparable dependence with C0(4,7-DSPphen)3~- has prompted further experiments, particularly as the acid dissociation pK, of 6.1 indicated by the previous data is not far removed from the pK, for cacodylic acid. The behavior observed in acetate buffer (pH 4.8-5.7) is in agreement with the cacodylate data (Figure 2 and Table II16). As a further check the p H of a number of protein solutions was adjusted to the final pH value (rather than being kept at p H 7.5 prior to mixing) without any observable change in rate constants. With collidine (Table III6) and phosphate (pH 7.5),8 rate constants are some 30 and 1 WO,respectively, higher than in cacodylate. Association of Co(pher1)3~+with PCul has been observed previously in kinetic studies a t p H 7.5, I = 0.10 M, and from a treatment as in (3) values K = 167 f 20 M-' and k,, = 17.9 f 1.7 s-l were reported.8 Information regarding the p H dependences of K and k,, has now been obtained. At p H 5.5, rate constants kobsd (Table III)I6 give a linear plot with [ c o (phen)j3+], Figure 3, from which K is estimated to be
