Bean plastocyanin

A solution structure of French Bean plastocyanin has been reported by Wright and co-workers,19 using nuclear magnetic resonance techniques described in Section 3.5 of Chapter 3. The structure, determined from a plastocyanin molecule in solution rather than in a solid-state crystal, agrees well with that of reduced poplar plastocyanin X-ray crystallographic structure reported above. Conformations of protein side chains constituting the hydrophobic core of the French bean plastocyanin are well-defined by the NMR technique. Surface side chains show... 

Fig. 1. The a-carbon chain structure of poplar plastocyanin [16] including details of the active site and the remote acidic patches 42-45 and 59-61. The first of these has been modified to include an acidic residue at position 45 as e.g. for spinach and French bean plastocyanins...

Binding at the remote site has also been detected in studies on the quenching of the excited states [Cr(phen)3] and [Ru(bipy)3] by French bean plastocyanin [103]. The model adopted allows for electron transfer from the remote and adjacent sites, where at low protein concentrations the adjacent pathway is 10 times faster. At the higher concentrations of protein, up to 4 X 10 M, an interesting feature is the evidence for an adduct in which two PCu(I) molecules are associated with one inorganic complex. The oxidant is believed to be sandwiched between two PCu(I) s. 

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... 

The other way to study the "conductivity of protein molecules towards electron tunneling is to investigate the quenching of luminescence of electron-excited simple molecules by redox sites of proteins [95,96]. Experiments of this sort on reduced blue copper proteins have involved electron-excited Ru(II)(bpy)3, Cr(III)(phen)3, and Co(III)(phen)3 as oxidants. The kinetics of these reactions exhibit saturation at protein concentrations of 10 3 M, suggesting that, at high protein concentrations, the excited reagent is bound to reduced protein in an electron transfer precursor complex. Extensive data have been obtained for the reaction of reduced bean plastocyanin Pl(Cu(I)) with Cr(III)(phen)3. To analyze quenching experimental data, a mechanistic model that includes both 1 1 and 2 1 [Pl(Cu(I))/ Cr(III)(phen)3] complexes was considered [96]... 

Research aimed at identifying the ligands comprising the flattened tetrahedral blue copper center has been particularly intense in the case of plastocyanin. Direct evidence for a sulfur ligand has come from x-ray photoelectron spectral (XPS) experiments on bean plastocyanin, where a large shift of the S2p core energy of the single cysteine (Cys-85) residue in the protein upon metal incorporation (164.5, apo 169.8, native 168.8 eV, Co(II) derivative) was observed (15). The two histidines in spinach plastocyanin exhibit pK values below 5 in NMR titration experiments,... 

Strong evidence for cysteine sulfur coordination in stellacyanin has been obtained (18) in XPS experiments. Thioether coordination is ruled out in this case, as the protein does not possess any methionine (6). It is probable that the other ligands are similar, but not necessarily identical, to those of bean plastocyanin. 

Other observations relevant to possible ligands to the Cu2+ are (a) None of the plastocyanins analyzed contains tryptophan or arginine, (b) The fluorescence of tyrosine in parsley plastocyanin is partially quenched but there is no evidence that t5nosine is bound to Cu2+. (c) Spinach and French bean plastocyanin contain two histidine residues while parsley plastocyanin appears to have only one. 

The kinetics of electron transfer reactions between spinach plastocyanin and [Fe(CN)6] ", [Co(phen)3] , and Fe(II) cytochrome c have been studied as a function of ionic strength. Applications of the equations of Van Leeuwen support the proposal of two sites of electron transfer, with [Co(phen)3] binding near residues 42-45 and the interaction of [Fe(CN)6] at a hydrophobic region near the copper ion. Pulse radiolysis has been employed to measure the rates of electron transfer from Ru(II) to Cu(II) in plastocyanins from Anabaena variabilis and Scenedesmus obliquus which have been modified at His-59 by [Ru(NH3)5] . The small intramolecular rates (<0.082 and <0.26 s , respectively) over a donor-acceptor distance of 12 A indicate that electron transfer from the His-59 site to the Cu center is not a preferred pathway. A more favorable route, via the acidic (residues 42-44) patch ( 14 A to Cu), is supported by the rate of >5 x 10 s for the reduction of PCu(II) by unattached [Ru(NH3)5im] . The intramolecular electron transfer from Fe(II) in horse cytochrome c to Cu(II) in French bean plastocyanin ( 12 A from heme edge to Cys-84 S), in a carbodiimide cross-linked covalent complex, proceeds with a rate of 1.05 x 10 s . The presence of the... 

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... 

The solution conformation of plastocyanin from French bean, spinach, and S. obliquus has now been determined from distance and dihedral angle constraints derived by NMR spectroscopy [37,40]. These two-dimensional NMR studies have indicated a well defined backbone conformation, which is very similar to that of poplar PCu in the crystalline state. However, in the case of S. obliquus there are deletions at positions S7 and 58 which influence the shape in the acidic region and in particular close to residues 59-61. The gap which is created is in effect repaired with consequent tightening of the loop 57-62 as indicated in Fig. 5. One of the pronounced bulges at the remote site of poplar and presumably other higher plant plastocyanins is not therefore present in S. obliquus (or plastocyanin from other green algae) [31, 32], as well as parsley... 

Reduction potentials (Eg) for different plastocyanins, the PCu(II)/PCu(I) couple, have been determined by spectrophotometric titration against, e.g. [Fe(CN)e] . At pH 7.5 for higher plant and green algal plastocyanins values are close to 370 mV at 25 °C, 1=0.10 M(NaCl) [1]. Thus French bean gives a value 360 mV and S. obliquus 363 mV [50]. However, A. variabilis gives an... 

Parsley, spinach, French bean, poplar and S. obliquus (but not A. variabilis) conform extensively to the above criteria for reaction at the remote site. There is extensive evidence for cytochrome f reacting at the remote site on plastocyanin. The aromatic residue at 83 would seem to be a prime candidate as lead-in group for electron transfer. Desolvation at the surface around 83, and interaction with an aromatic component on the reaction partner, e.g. the porphyrin ring of cytochrome f, may be important. The exact manner of electron transfer has yet to be confirmed. The distance from the aromatic ring of Tyr83 to the Cu for electron transfer is 12 A. 

PCY Plastocyanin Phaseolus vulgaris (French bean) Moore fl/., 1991... 

The first crystal structure information on a blue copper protein, for poplar plastocyanin in the Cu(II) state, was published in 1978 (2, 3). Since then, the Cu(I) state and related apo and Hg(II) substituted forms (5, 6), the green algal plastocyanin from Enteromorpha prolifera [Cu(II)] (7), azurin from Alcaligenes denitrificans [Cu(II) and Cu(D] (8, 9), azurin from Pseudomonas aeruginosa [Cu(II)] (10, 11), as well as pseudoazurin from Alcaligenes faecalis S-6 (12), and the cucumber basic protein, both in the Cu(II) state, have been published (13), making this one of the best-documented class of proteins. In addition, information as to three-dimensional structure in solution has been obtained from two-dimensional NMR studies on French bean and Scenedesmus obliquus plastocyanins (14,15). This review is concerned in the main with the active site chemistry. Other recent reviews are listed (16-20). 

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