Plastocyanin rate constants

Rate Constants and Reactivity. Electron-transfer reactions of plastocyanin (and other metalloproteins) are so efficient that only a narrow range of redox partners (having small driving force) can be employed. Rates are invariably in the stopped-flow range, Table I. Unless otherwise stated parsley plastocyanin... 

Summary of reduction potentials and rate constants for reactions with plastocyanin PCu(I) and PCu(II) at 25<>C, pH 7-8,... 

Figure 3. Dependence of first-order rate constants ko6, (25 °C) vs. [Co(phen)s3+] for the oxidation of plastocyanin PCu(I). Conditions pH, 7.5 (phos) and I, 0.10 M (NaCl). Key , spinach and A, parsley. (Reproduced from Ref. 10. Copyright 1978, American Chemical Society.)...

Figure 6. The variation of rate constants k (25 °C) with pH for the reaction of Fe(CN)64 with parsley plastocyanin PCu(II) [I = 0.10 M (NaCI)]. Key A, acetate and O, cacodylate. (Reproduced from Ref. 10. Copyright 1978, American...

Figure 8. The blocking effect (25 °C) of redox inactive complexes on the reaction of parsley plastocyanin PCu(I) + Co(phen)s3 Rate constants were determined at pH 7.5 (for U and m) and pH 5.8 (for A) [I = 0.10 M (NaCl)].

Reaction with Cr(III) Modified Plastocyanin. From thermo-lysin proteolysis experiments Farver and Pecht (20) have concluded that reduction of PCu(II) with labelled Cr0 20)52+ (1 1 mole amounts) at pH 7 gives a product in which Cr(III) is attached to the peptide chain 40-49. Coordination of the Cr at one or two carboxylates in the 42-45 patch is favoured. It has now been demonstrated that rate constants (25oC) for the reaction of PCu(I).Cr(III) + Co(phen)33+ are decreased by 16%. 

Other Studies. Experiments in which rate constant pH profiles, blocking effects of redox inactive complexes as well as the effect of Cr(III) modification should now be possible enabling sites on plastocyanin used by cytochrome f and P700 to be specified (25,26). 

Table 2. Acid dissociation pK values, 1 = 0.10 M(NaCl), relating to the active site protonation of different plastocyanins, PCu(I), as determined by (a) proton NMR (b) the variation of rate constants (25 °C) with pH for the [FelCN) ] oxidation of PCu(I), 1 = 0.10 M(NaCl), and (c) similar experiments with [Co(phen)3] " as oxidant. The latter is an apparent value only, and is believed to be composite due to reaction occurring at the remote site...

Earlier suggestions that the two uncoordinated and invariant residues His35 (inaccessible to solvent and covered by polypeptide) and His83 (remote and 13 A from Cu) are, from effects of [H ] on rate constants (and related pKg values), sites for electron transfer may require some re-examination. Thus, it has been demonstrated in plastocyanin studies [50] that a surface protonation can influence the reduction potential at the active site, in which case its effect is transmitted to all reaction sites. In other words, an effect of protonation on rate constants need not necessarily imply that the reaction occurs at the site of protonation. His35 is thought to be involved in pH-dependent transitions between active and inactive forms of reduced azurin [53]. The proximity of... 

Table 4. Comparison of rate constants (25 °C) for the reactions of parsley and spinach plastocyanins, pH7.5,1=0.10 M(NaCl) [95, 99, 100]...

These have been determined for the [Fe(CN) ] oxidation of parsley plastocyanin, and at pH7.5, I=0.10M(NaCl), are AHJ = — 3.3 kcalmol and AS = —47 cal K mol" [39]. To account for the negative enthalpy term it has been suggested that the second-order rate constant is composite incorporating... 

There are surprisingly few examples and considerable care is required. A number of earlier reports have been checked out [90,95,108], and shown to be incorrect with effects certainly not as extensive as claimed. The first plastocyanin example (1978) was the [Co(phen)3] oxidation of parsley PCu(I) [39]. In this study first-order rate constants (k bs) obtained with [Co(phen)3] in > 10-fold excess of PCu(I) ( 10 M) give a non-linear dependence on [Co(phen)3 ] (concentrations of oxidant to 3 x 10 M), Fig. 7. Such behavior can be accounted for by the reaction sequence (2)-(3),... 

Fig. 8. Competitive inhibition of redox inactive complexes (I) on the [Co(phen)j] oxidation of parsley plastocyanin PCu(I) [98]. Second-order rate constants (25 °C), shown as relative values, were determined at pH 5.8 (Mes), 1=0.10 MfNaO) with [Pt(NH3)6] ( ), [(NH3)5CoNHj(NH3)5]s] [Co (III)4]a (A) and [CoflllUg ( ). Full formulae of the latter two complexes are as indicated above...

The kinetics of the reactions of horse cytochrome c(II), M, 12,400, (charge 8+) reduction potential 260 mV, with parsley and French bean plastocyanins PCu(II) (charges — 7 and — 8 respectively), have been studied. As in the case of HIPIP, cytochrome c is not a physiologically relevant protein. It is nevertheless important in assessing different approaches prior to investigating the reactions of physiological redox partners. In the case of the reaction of parsley PCu(II) with cytochrome c(II), the rate constant (25 °C) is 1.5 X 10 s at pH7.6, 1 = 0.10 M(NaCl) [141]. There is no evidence... 

Cryokinetic studies of the plastocyanin-ferricyanide redox reactions in 50 50 v/v MeOH + H2O, pH = 7.0, p = 0.1 M reveal an Eyring plot shown for the second-order rate constant k from 25 °C to -35°C. The reaction is irreversible over the whole temperature range and there is no evidence for a change in the Cu(I) active site. Recalling that these reactions may involve consecutive steps, explain the deviation from a linear Eyring plot. F. A. Armstrong, P. C. Driscoll, H. G. Ellul, S. E. Jackson and A. M. Lannon, J. Chem. Soc. Chem. Communs. 234 (1988). 

More subtle factors that might affect k will be the sites structures, their relative orientation and the nature of the intervening medium. That these are important is obvious if one examines the data for the two copper proteins plastocyanin and azurin. Despite very similar separation of the redox sites and the driving force (Table 5.12), the electron transfer rate constant within plastocyanin is very much the lesser (it may be zero). See Prob. 16. In striking contrast, small oxidants are able to attach to surface patches on plastocyanin which are more favorably disposed with respect to electron transfer to and from the Cu, which is about 14 A distant. It can be assessed that internal electron transfer rate constants are =30s for Co(phen)3+, >5 x 10 s for Ru(NH3)jimid and 3.0 x 10 s for Ru(bpy)3 , Refs. 119 and 129. In the last case the excited state Ru(bpy)3 is believed to bind about 10-12 A from the Cu center. Electron transfer occurs both from this remote site as well as by attack of Ru(bpy)j+ adjacent to the Cu site. At high protein concentration, electron transfer occurs solely through the remote pathway. 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Fe "-OOH (ES) complex, 43 95-97 heme-bound CO, 43 115 lock-and-key model, 43 106-107 mutation in proximal heme cavity, 43 98 residue location, 43 101-102 van der Waals surfaces, 43 112-113 Velcro model, 43 107 zinc-substituted, 43 110-111 plastocyanin, cross-linked, cyclic voltammogram, 36 357-358 promoters, 36 345-346 protein-electrode complex, 36 345, 347 redox potential, 36 349 self-exchange rate constants, 36 402 stability at electrode/electrolyle interface, 36 349-350... 

There are many instances for azurin and plastocyanin where limiting kinetic behaviour is observed, and attributed to the formation of an adduct between the protein and the inorganic complex followed by electron transfer. Values of the association constants and of the electron-transfer rate constants may then be calculated. This situation has not been observed in the case of stellacyanin, which differs from azurin and plastocyanin in that it has an overall positive charge at pH 7 (of +7 in the case of the reduced protein). The electron-transfer rate constants are often associated with fairly large negative values for the entropy of activation (in the range -84 to -210 J K1 mol-1), which are not expected for electron transfer within a compact assembly. 

The results of kinetic studies of the reduction of stellacyanin, plastocyanin, and azurin by Fe(EDTA)2" are summarized in Table III (20, 21). The order of cross reaction rate constants (ki2 values) is stellacyanin > plastocyanin > azurin, which is surprising, as considerations based on driving force alone would predict stellacyanin to be the least reactive of... 

Unfortunately, experimental kn values are not available for any one of the three blue proteins. It has been established, however, that the self-exchange rates for azurin (24) and plastocyanin (25) are both slow on the NMR time scale at 25°. It is not likely that the predicted kn values from the Fe(EDTA)2 reactions will correspond to the measured self-exchange rate constants. Indeed, agreement between calculated and measured kn values is only to be expected if the activation requirements of both partners in a cross reaction are exactly the same as those used in their respective self-exchanges. Access to a buried, or at best partially exposed, outer-sphere redox center should vary substantially for different reactants, and it is likely that calculated kn values will span a wide range in such cases. 

In this complex, there are two optically active sites. Spinach plastocyanin is a type I copper protein, in which two reactive sites have been identified on its surface, at least. The electron transfer reaction occurs with significantly large stereoselectivity the ratio of the observed reaction rate constant (k /k ) is 1.6 to 2.0. The difference in the activation enthalpy, AAH a, is 3.0 kJ mol-1, and the difference in the activation entropy, AS (a-a) is 15 J mol-1 K-1. This means that the stereoselectivity arises from the entropy term. 

Rates of Cu+ to Ru + electron transfer also have been measured in modified mutants of spinach plastocyanin, a blue copper protein from the photosynthetic ET chain [79], Ru-bipyridine complexes were introduced at surface sites, with Cu-Ru distances ranging from 13 to 24 A. ET rate constants, measured using laser flash-quench techniques, vary from 10" to 10 s. ET in Ru-modified plastocyanin is not activationless as it is in Ru-modified azurin, suggesting a slightly greater reorganization energy for the photosynthetic protein. The distance dependence of ET in Ru-modified plastocyanin is exponential with a distance decay factor identical with that reported for Ru-modified azurin (1.1 A ). 

Figure 1. Ionic strength dependence of observed rate constants for ET from two reduced cytochromes (c555 and C551) to oxidized plastocyanin. Solid lines are theoretical fits to the data points using the parallel plate model (only the V term is included).

The reduction potentials of different plastocyanins increase as the pH is decreased below 7 (Fig. 8), due to protonation of His 87 at the Cu(I) active site and resultant redox inactivation. Much of this information has been obtained from rate constants (18, 57, 58). The reduction potential of the basic A. variabilis plastocyanin is noticeably smaller than for plastocyanins from higher plant and green algal... 

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