Plastocyanin acidic residues

Figure 1. Structure of plastocyanin (2) showing the positions of a-carbon atoms of amino acid residues. The active site and positions of the conserved (plant) negative patch (42-45) and Tyr 83 are indicated (%).

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...

The subunits are arranged in the crystals as homotetramers with D2 symmetry. The structure of a subunit is shown schematically in Fig. 1 (87). Each subunit of 552 amino acid residues has a globular shape with dimensions of 49 x 53 x 65 A and is built up of three domains arranged sequentially on the polypeptide chain, tightly associated in space. The folding of all three domains is of a similar jS-barrel type. It is distantly related to the small blue copper proteins, for example, plastocyanin or azurin. Domain 1 is made up of two four-stranded jS-sheets (Fig. lb), which form a jS-sandwich structure. Domain 2 consists of a six-stranded and a five-stranded jS-sheet. Finally, domain 3 is built up of two five-stranded jS-sheets that form the jS-barrel structure and a four-stranded j8-sheet that is an extension at the N-terminal part of this domain. A topology diagram of ascorbate oxidase for all three domains and of the related structures of plastocyanin and azurin is shown in Fig. 2. Ascorbate oxidase contains seven helices. Domain 2 has a short a-helix (aj) between strands A2 and B2. Domain 3 exhibits five short a-helices that are located between strands D3 and E3 (a ), 13 and J3 (a ), and M3 and N3 (a ) as well as at the C terminus (ag and a ). Helix 2 connects domain 2 and domain 3. 

Homologies between azurin and plastocyanin have been considered. A schematic representation of the polypeptide folding of A. denitrifi-cans azurin is shown in Fig. 4. Essential differences as compared to plastocyanin are the prominent 52-81 azurin flap, including an a-helix section (Fig. 5), which replaces the acidic residues 42-45 on plastocyanin. There is an extension of the plastocyanin 33-37 section, and the 59-61 acidic residues are also replaced. 

The remote site on plastocyanin consists of acidic residues 42-45 and 59-61 on either side of Tyr 83. Typically, positively charged complexes react —50% at this site. Evidence has been obtained for cytochrome c (97) and cytochrome f(35) reacting more extensively (possibly exclusively) at this site. 

The electron-transfer kinetics of PSI-200 particles cross-linked with wild-type and mutant plastocyanins were examined by flash-induced absorbance changes. PSI-200 particles cross-linked with wild-type PC, shown in Fig. 8, trace (a), displays two P700 re-reduction phases with ty, of 13 jus and 141 /js, respectively, comparable to those found in vivo. For PSI-200 cross-linked with a mutant PC with modified residues 42 to 45 in the southern acidic patch, i.e., Asp-42->Asn, Glu-43->Gln, Asp-44->Asn and Glu-45->Gln, the rate of P700 re-reduction is dramatically retarded, as shown in Fig. 8, trace (b), giving two kinetic phases with ty of 160 //y and 766 /zy. When a mutant PC in which only one residue in the northern acidic patch was mutated, i.e., Glu-59->Gln, the resulting complex with PSI-200 yielded flash-induced P700 re-reduction kinetics which were almost the same as that for the wild-type plastocyanin, with ty, values of 13 /zy and 149 /js, as shown in Fig. 8, trace (c). When all three acidic residues in the northern acidic patch were converted to neutral ones, i.e., Glu-59->Gln, Glu-60->Gln and Asp-6l->Asn, the rapid re-reduction phase still retained a ty, of 14 /zy [Fig. 8, trace (d)]. 

Reduction of plastocyanin by cytochrome / would presumably involve some direct interaction between them. Cyt/will be described in detail in Chapter 35, but some relevant information on the composition and structure of Cyt / is given here to provide a perspective on the PC/Cyt / interaction. Cyt /, a subunit of the Cyt complex, is a c-type cytochrome of 33 kDa molecular mass with an in situ redox potential of -370 mV. The amino-acid composition has been determined for Cyt/from several species, that for turnip being shown in Fig. 11. Turnip Cyt/, containing 252 amino-acid residues, was crystallized and its three-dimensional structure determined in 1994 by Martinez, Huang, Szczepaniak, Cramer and Smith " at 2.3 A resolution, now refined to 1.96 A (see Chapter 35). 

In the meantime, Morand, Frame, Colvert, Johnson, Krogmann and Davis used EDC to cross-link spinach PC and turnip Cyt/to form an adduct with a 1 1 stoichiometry. The authors found that Lys-187 of Cyt/cross-linked with Asp-44 of PC, and another, but unidentified, amino-acid residue of Cyt/cross-linked with Glu-60 of PC. Interestingly, the authors found that the PC/Cyt/adduct was incapable of binding with photosystem I or donating electrons to it. This latter finding is apparently consistent with other evidence that Asp-44 is one of the group of four acidic residues (42-45) that are generally believed to bind PsaF and crucial for electron transfer between plastocyanin and photosystem I. 

The amino acid residues coordinating the copper ion occupy corresponding positions in the /3-sheets of plastocyanin, pseudoazurin, and amicyanin (Table 5). 

Until now, pseudoazurin has only been found in bacteria, e.g., in the denitrifying bacteria Alcaligenes faecalis and Achromobacter cycloclastes. It is the electron donor of the green copper-protein nitrite reductase which catalyses the reduction of nitrite (NO2) to nitrogen monoxide (NO) [86-89]. The physiological electron donor of pseudoazurin is as yet unknown [70]. Pseudoazurin has a molecular mass of approximately 13.5 kD and a chain length of about 123 amino acid residues [88]. The additional amino acids, as compared with plastocyanin, form the C-terminal end of the protein (Fig. 14). The pseudoazurins have redox potentials of about 230 mV [90,91]. 

Amicyanins function as electron carriers in the respiratory chains of some me-thylotrophic bacteria, e.g., Thiobacillus versutus [92]. They transfer single electrons from methylamine dehydrogenase to a cytochrome c [78] which then transfers the electron to cytochrome c oxidase. Amicyanin from Pseudomonas denitrificans has a molecular mass of 11.6 kD and contains 106 amino acid residues. Amicyanin contains one /J-sheet more than the eight of plastocyanin and pseudoazurin, the result of several additional amino acids at its N-terminus [78] (Fig. 15). Like pseudoazurin, amicyanin is found exclusively in bacteria. 

Blue copper proteins contain a minimum of one Type 1 Cu centre, and those in this class include plastocyanins and azurins. Plastocyanins are present in higher plants and blue-green algae, where they transport electrons between Photosystems I and II (see above). The protein chain in a plastocyanin comprises between 97 and 104 amino acid residues (most typically 99) and has 10 500. Azurins occur in some bacteria and are involved in electron transport in the conversion of [N03] to N2. Typically, the protein chain contains 128 or 129 amino acid residues (M 14600). 

Figure 2-21. Structure of poplar plastocyanine. O represents the a-carbons of amino acid residues.

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... 

Determination of ihe location of the label. The location of the CDNB moiety was determined for peaks F2, F3, F4 and F6. Each san4>1e was treated with trypsin and the tryptic peptides were separated using reverse phase HPLC and eluted with a 9 1 acetonitrile/water gradient. In general, CDNB modification causes the loss of a tryptic cleavage site which results in the loss of two tryptic peptides accompanied by the appearance of a new tryptic peptide at another location. In each case, the new tryptic peptides were subject to amino acid analysis and sequencing of the first five amino acid residues. Each modified form was labelled in a different location on the plastocyanin molecule. The locations are shown in Table I and Fig 1. 

In summary, there are two classes of amino acid residues one involved in critical alignment of docking for electron transfers and the other involved in crude orientation or transient complex formation. Those involved in critical docking aUgnment tend to be different for the two substrates, Fd and those bound to opposite sides of FNR. Finally, hydrophobic clusters usually line the binding cavity and are critical for electron transfer. The situation with plastocyanin and cytochrome Cg to be presented next is similar the two independent sets of data demonstrate a common design scheme for redox partners of long-distance electron transfers. 

From 13 completed amino-acid sequences and 54 partial sequences (>40 residues) of plastocyanins from higher plants it appears that sixty residues are invariant and 7 are conservatively substituted 02,7). With three algal plastocyanins included there are 39 invariant or conservatively substituted groups. It is believed that the same structural features apply to the whole family, and that highly conserved residues are an indication of functional sites on the protein surface. The upper hydrophobic and right-hand-side surfaces are believed to be particularly relevant in this context, the latter including four consecutive... 

Fig. 3. Plastocyanin amino-acid sequences relevant to this review. Other sequence information is included. There are now 24 known sequences (18 formerly in Ref. [ I]). The 23 residues invariant throughout all 24 known higher plant and algal plastocyanin sequences are indicated ( ), 5 others (A) are invariant if A. variabilis is excluded, and a further 19 (o) if only the higher plant sequences are considered. Eieletions are indicated ( ), and residues which coordinate the Cu(V)...

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... 

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