Plastocyanin acidic regions

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... 

A transit peptide consisting of a hydrophobic 66 amino acid long peptide interspersed with positively charged residues has been identified and sequenced [52]. This is initially attached to the 99 amino acids of the mature plastocyanin, and is responsible for taking the plastocyanin across membranes into the thylakoid region of the chloroplast. 

There are no uncoordinated histidine residues in higher plant plastocyanins. However, some algal plastocyanins, including S. obliquus and A. variabilis have a histidine at position 59, which is of particular interest since this lies within the remote acidic patch region [132]. Differences between the two are that whereas S. obliquus PCu(I) is acidic (charge —9), A. variabilis is basic (charge 1-I-). Also S. obliquus has deletions at 57 and 58 with a consequent tightening of the peptide chain in this locality. Fig. 5. Both these plastocyanins were therefore Ru-modified. 

Fig. 1. Ribbon drawing of the cyanobacterium Synechocystis sp. PCC 6803 Cu(II)-plastocyanin structure showing the secondary structure elements of the protein. The metal ion is represented as a sphere of arbitrary dimensions (Bertini et al., 2001b). The letters N, E, S, and W refer to the cardinal points. The so-called acidic patch present in the structure of plant plastocyanins is located in the E region.

Plastocyanin contains two copper atoms per molecule. Plastocyanin in photosystem I is soluble in the lumen region and can be readily released in a hypotonic medium and purified by DEAF chromatography. In the oxidized form the copper protein is blue with a major absorption band at 597 nm = 4.7 mM two weaker absorption bands at 460 and 770 nm, and one band with vibrational structure characteristic of amino acids in the ultraviolet region (see Fig. 2). Plastocyanin in the reduced state does not absorb in the visible region. The low-temperature EPR spectrum of oxidized PC has characteristic g-values at 2.05 and 2.23. 

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... 

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