Fuminori Yoshizaki

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera☆

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.

Spectroscopic characterization and kinetic studies of a novel plastocyanin from the green alga Ulva pertusa

Abstract A novel plastocyanin from the green alga Ulva pertusa was isolated and characterized. The electronic absorption and the electron paramagnetic resonance spectroscopic properties of Ulva plastocyanin showed features characteristic of the usual plastocyanins reported so far. However, the resonance Raman spectrum on excitation at 607 nm indicated the Raman band of Cu-SCys at 413 cm−1; this is at a lower frequency than the corresponding Raman band of higher plant plastocyanins. Electron-transfer reactions were investigated with [Fe(CN)6]3− and [Co(phen)3]3+ complexes. The electron-transfer rate constant of Ulva plastocyanin was determined to be (1.18 ± 0.06) × 105 M−1 s−1 for the reaction with [Fe(CN)6]3− at pH 7.5, and the intramolecular electron-transfer rate constant and equilibrium constant for complex formation for the reaction with [Co(phen)3]3+ were evaluated to be 7.7 ± 0.6 s−1 and (4.2 ± 0.4) × 102 M−1 respectively. The electron transfer rate constant for the reaction with [Fe(CN)6]3− at pH 7.5 is two times larger than the values obtained from the other higher plant plastocyanins. The kinetic behavior suggested that the structure is slightly different from those of other plastocyanins. It has been reported that the electron-transfer reaction of plastocyanin is inhibited by the protonation of the active site histidine (His87 in poplar plastocyanin) at acidic pH. The dependence on pH of the rate constant for the reaction with [Fe(CN)6]3− was investigated. The acid-dissociation constant accompanying the electron-transfer reaction was determined to be pKH = 5.8. Second-order rate constants for the reduction of plastocyanin by cytochrome c were determined to be (1.71 ± 0.04) × 106 M−1 s−1 at I = 0.1 M (NaCl), pH 7.0 (20 mM Tris—HCl buffer). The saturation kinetic behavior for the reaction was not observed even at the lower ionic strength (20 mM Tris—HCl).

Preliminary crystallographic data for plastocyanins from an alga (Enteromorpha prolifera) and from cucumber (Cucumis sativus)

The plastocyanins from a green alga (Enteromorpha prolifera) and cucumber (Cucumis sativus) have been crystallized. Crystal data are as follows: E. prolifera plastocyanin, space group I4, a = b = 53.9 A, c = 59.4 A, Z = 8; C. sativus plastocyanin, space group P4(1) (or P4(3) ), a = b = 66.7 A, c = 46.0 A, Z = 8. Accordingly, the asymmetric units of the crystals contain one and two molecules, respectively.