Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability.

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

Twin plastocyanin dimorphism in tobacco.

Two iso-plastocyanin fractions, oxidized b-plastocyanin, PCb(II) and reduced a-plastocyanin, PCa(I), have been isolated from whole tobacco leaves by conventional chromatography on DEAE-cellulose. The isoelectric points of PCa and PCb at 10 degrees C were found to be 3.99 and 3.97, respectively. When the primary structures were analysed, a microheterogeneity within both PCa and PCb was observed. By appropriate peptide arrangements the amino-acid sequences of two PCa (PCa' and PCa") and two PCb (PCb' and PCb") have been differentiated. All four sequences contain 99 amino-acid residues. PCa' and PCa" differ in one position, where Ser-58 in PCa' is replaced by Pro in PCa".PCb' and PCb" differ in three positions, where Gly-65, Thr-81 and Ala-85 in PCb' are replaced by Ala, Ser and Ser in PCb", respectively. PCa (PCa'/PCa") generally differs from PCb (PCb'/PCb") in three positions, where Val-52, Glu-61 and Tyr-62 in PCa'/PCa" are replaced by Ala, Asp and Leu in PCb'/PCb", respectively. Fluorescence spectra of oxidized tobacco PCa and PCb have been characterized with an emission-maximum position at around 340 nm. The presence of one extra tyrosyl (Tyr-62) in PCa results in a weak increase of the maximal intensity in conjunction with a slight blue-shift of the maximum position.

Microheterogeneity of parsley plastocyanin.

A procedure for isolation of two iso-plastocyanins from parsley has been described here. Three consecutive chromatographic steps on DE-52-Whatman cellulose were applied for isolation of two total plastocyanin (PC) fractions, oxidized [PC(II)] and reduced [PC(I)]. By chromatofocusing of PC(II) on Polybuffer exchanger 74 two different plastocyanins, designated as plastocyanin a (PCa) and plastocyanin b (PCb), were obtained. The isoelectric points (pI) of PCa and PCb at 10 degrees C are 4.16 and 4.14, respectively. The complete amino acid sequences of PCa and PCb were determined. The two iso-proteins consist of 97 amino acid residues and differ only at sequence position 53, where Glu in PCa is replaced by Asp in PCb.