SPECTROSCOPIC CHARACTERIZATION AND KINETIC-STUDIES OF A NOVEL PLASTOCYANIN FROM THE GREEN-ALGA ULVA-PERTUSA

A novel plastocyanin from the green alga Ulva pertusa was isolated and characterized. The electronic absorption and the electron paramagneti c resonance spectroscopic properties of Ulva plastocyanin showed featu res characteristic of the usual plastocyanins reported so far. However , the resonance Raman spectrum on excitation at 607 nm indicated the R aman band of CuSCys at 413 cm(-1); this is at a lower frequency than t he corresponding Raman band of higher plant plastocyanins. Electron-tr ansfer reactions were investigated with [Fe(CN)(6)](3-) and [Co(phen)( 3)](3+) complexes. The electron-transfer rate constant of Ulva plastoc yanin was determined to be (1.18 +/- 0.06) x 10(5) M-1 s(-1) for the r eaction with [Fe(CN)(6)](3-) at pH 7.5, and the intramolecular electro n-transfer rate constant and equilibrium constant for complex formatio n for the reaction with [Co(phen)(3)](3+) were evaluated to be 7.7 +/- 0.6 s(-1) and (4.2 +/- 0.4) x 10(2) M-1 respectively. The electron-tr ansfer rate constant for the reaction with [Fe(CN)(6)](3-) at pH 7.5 i s two times larger than the values obtained from the other higher plan t plastocyanins. The kinetic behavior suggested that the structure is slightly different from those of other plastocyanins. It has been repo rted that the electron-transfer reaction of plastocyanin is inhibited by the protonation of the active site histidine (His87 in poplar plast ocyanin) at acidic pH. The dependence on pH of the rate constant for t he reaction with [Fe(CN)(6)](3-) was investigated. The acid-dissociati on constant accompanying the electron-transfer reaction was determined to be pK(H) = 5.8. Second-order rate constants for the reduction of p lastocyanin by cytochrome c were determined to be (1.71 +/- 0.04) x 10 (6) 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). (C) 1998 Elsevier Science S.A. All rights reserved.