Plastocyanin reactivity

Structure and electron transfer reactivity of the blue copper protein, plastocyanin. A. G. Sykes, Chem. Soc. Rev., 1985,14, 283 (117). 

Metalloproteins fall into three main structure categories depending on whether the active site consists of a single coordinated metal atom, a metal-porphyrin unit, or metal atoms in a cluster arrangement. In the context of electron-transfer metalloproteins, the blue Cu proteins, cytochromes, and ferre-doxins respectively are examples of these different structure types. Attention will be confined here mainly to a discussion of the reactivity of the blue Cu protein plastocyanin. Reactions of cytochrome c are also considered, with brief mention of the [2Fe-2S] ferredoxin, and high potential Fe/S protein [HIPIP]. 

It is timely to review the reactivity of plastocyanin in the light of recent aqueous solution studies, and the elegant structural work of Freeman and colleagues on both the PCu(I) and PCu(II) forms (1 2) Plastocyanin now ranks alongside cytochrome c (3) as the electron-transfer metalloprotein for which there is most structural information. 

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... 

There have been many studies on the reactivity of plastocyanin. Two sites (or regions) have been identified on the surface of the molecule as relevant to electron transfer. One is the adjacent site at or near His87, and the other a more... 

While there is at present no full understanding as to why plastocyanin should require two sites for reaction, there is now much evidence detailing this two-site reactivity. Moreover, the recent X-ray crystal structure of ascorbate oxidase (which has 4 Cu atoms per molecule) has indicated a plastocyanin-like domain, with the two type 3 Cu s (in close proximity with the type 2 Cu) located at the remote site. Fig. 2 [5]. Since electrons are transferred, from the type 1 Cu to O2 bound at the type 3 center this structure defines two very similar through-bond routes for biological electron transfer. 

Much of this review has been devoted to plastocyanin. Evidence has been presented in support of plastocyanin electron transfer reactivity at adjacent and remote sites. Of the two, reactivity at the remote site is of particular interest in the... 

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... 

Based on the Fe(EDTA)2- results, the blue copper center in stellacyanin appears to be much more accessible than that situated in either azurin or plastocyanin. Thus it should be profitable to compare the electron transfer reactivities of these three proteins with a variety of redox agents. Kinetic studies of the oxidation of the three blue proteins by Co(phen)33+ have been made (26), and the results together with those for other redox agents are set out in Table IV. The electrostatic corrections to the predicted kn values are modest both for the large charge on plastocyanin and the small one on azurin, as the protein selfexchange and the cross reaction work terms compensate. The reactivity... 

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. 

The structure of plastocyanin is known at a highly refined level, which allows interesting hypotheses on which part of the molecule is involved in interactions permitting electron transfer [73]. Several areas on the surface of the molecule have been modified with chemical reagents, which can change the binding and reactivity [74], which are highly sensitive to electrical interactions, as shown by the influence of cations on the rate of electron transfer (see e.g. Refs. 68 and 75). 

Once metals have been transported to their target tissue, they need to be distributed within the subcellular compartments where they are required, and need to be safely stored when they are in excess. Nearly 90% of Fe in plants is located in the chloroplasts, where it is required in the electron transfer chain, and in the synthesis of chlorophylls, haem, and Fe—S clusters. Fe, Cu, and Zn are also required in chloroplasts as cofactors for superoxide dismutases to protect against damage by reactive oxygen species during chloroplast development, and Cu is also required in other enzymes including the essential Cu protein plastocyanin. Pathways of intracellular metal transport in plant cells are illustrated in Fig. 8.10. Transport into the chloroplast is best characterised for Cu,... 

S Modi, M Nordling, LG Lundberg, Hansson and DS Bendall (1992) Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin. Biochim Biophys Acta 1102 85-90... 

The X-ray crystal structure of plastocyanin has recently been established (10), which indicated that the core of the molecule is hydrophobic and notably aromatic because six of the seven phenylalanine residues are clustered there. Polar side chains are distributed on the exterior of plastocyanin molecule. Many hypotheses have been proposed to explain the electron-transfer pathway to and from the metal center of plastocyanin, such as a tunnelling mechanism along hydrophobic channels (11). High reactivity and entropic favorability have been reported for the electron-transfer reaction of plastocyanin with Fe(II) complex (12). The Cu complex bound to the amphiphilic block copolymer is interesting as a metal compound of plastocyanin, because both polymer and apoprotein environments are considered to produce a hydrophobic environment and a large effect on the electron-transfer reaction through its entropic contribution. 

Similar results are noted for azurin where, for the reduced protein ACu, different binding sites witlj pATa values 7.6 ([Co(phen)3] +), 7.1 ([Fe-(CN)e] ), and no pH dependence ([Co(4,7-DPSphen)3] ) are reported. No dramatic switch-off in reactivity, as with plastocyanin, is noted, however, and the trend in the rates with protonation reflects that expected for the effect of electrostatic attraction on substrate binding. The pATa of 7.1 for [Fe(CN)8] reaction is shifted to pATa 6.1 for [Fe(CN)6] reduction of ACu and, by comparing the kinetic pATa values with those obtained by n.m.r., tentative assignments to histidines-35 and -83, which are not bound to the copper ion, are made. 

The kinetics of oxidation of Pseudomonas aeruginosa azurin, bean plastocyanin, and Rhus v. stellacyanin by the tris-cobalt(iii) complexes of phen and three of its derivatives have been reported (Table 15) the reactivity order for Co(phen)3 + as the oxidant (stellacyanin > plastocyanin > azurin) matches that found previously for the Fe(edta) reduction of the proteins (and for which ionic strength and pH effects have now been reported). It is suggested that the activation parameters for electron transfer from reduced plastocyanin and azurin may be accounted for in terms of oxidant-induced protein structural changes which expose active sites that are, by comparison with stellacyanin, inaccessible to reagent attack. Segal and Sykes have extended the work with plastocyanin and Co(phen)3 + to higher concentrations (up to 4.0 X 10 mol of oxidant and have observed a deviation from linearity in the... 

Since the replacement of Fe(II) by Zn(II) does not affect either the conformations or its association properties with other proteins, Zncyt(II) has been used to probe the photoredox reactions between cytochrome and plastocyanin. These studies show that the excited state potential of the cytochrome can influence the site of reactivity on plastocyanin, and that viscosity effects can be used to determine conformational changes that occur in the electron transfer process. " ... 

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