Anthony A. Donchev

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°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.

Complete amino acid sequence of poplar plastocyanin b

A second type of plastocyanin, plastocyanin b (PCb) was isolated from leaves of poplar Populus nigra var. Italica. The complete amino acid sequence of 99 residues in the single polypeptide chain of plastocyanin b has been determined: DVLLGADDG SLAFVPSEFS GEKIVFK NNAGFPHN FDED PSGV D SKISMSEE DLLNAKGETF EVALS KGEY FYCSPHQGA GMVGKV VN The sequence obviously demonstrates, that PCb, in comparison with the known plastocyanin, plastocyanin a (PCa), has 12 amino acid replacements (underlined letters): Ile 1→Val, Ile 21‐Ser 22‐Pro 23→Val‐Pro‐Ala, Ile 39‐Val 40→Val‐Leu, Ser 45‐Ile 46→ Ala‐Val, Ala 52→Val, Asn 76→Asp, Ser 81→Thr and Thr 97→Ile. The replacements at positions 40 and 76 of PCb are probably essential for differences in its redox and electrochemical properties, respectively.

Structural comparison of the poplar plastocyanin isoforms PCa and PCb sheds new light on the role of the copper site geometry in interactions with redox partners in oxygenic photosynthesis.

Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.

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.

Some physicochemical peculiarities of poplar plastocyanins a and b.

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectro photometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients ε597, equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0′: PCa: ε597 = (4.72 ± 0.08) mM-1 cm-1, Keq = 0.133 ± 0.009, E0′ = (354 ± 11) mV; PCb: ε597 = (5.23 ± 0.16) mM-1 cm-1, Keq = 0.175 ± 0.010, E0′ = (363 ± 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0′ for PCb is constant in the pH range 6.5 - 9.5, but decreases in the range 4.8 - 6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0′ in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50 - 60 mV per pH unit. It has been assumed that the weaker pH dependence of E0′ of PCb accounts for some structural differences between PCa and PCb

Changes in the content of poplar isoplastocyanins a and b during vegetation cycle

Changes in the content of isoplastocyanins a (PCa) and b (PCb) in Populus nigra leaves were studied during complete vegetation cycle (from April 30 to October 28). Measurements were made every 7 days. The procedure included extraction and purification of total protein followed by analytical isoelectric focusing and densitometry of obtained isophoregrams. The positions of PCa and PCb were determined by comparison with the positions of the highly purified two isoforms. The PCb/PCa ratio was calculated from the areas of the corresponding densitometric peaks. The amount of PCb was one half of PCa amount in the earliest stage of the leaf formation as well as during the longer part of the summer, e.g., the second half of July, August, and September. During the most active period of the poplar growth, e.g., June and the first half of July, the ratio was about or above unity. This result could be important for understanding the specific functional significance of PCa and PCb.

Extra- and intra-cellular lytic effects of Cytophaga sp. LR2 on the red microalgae Rhodella reticulata.

Aims: To evaluate the lytic activities of crude enzymes from Cytophaga sp. LR2 on Rhodella reticulata cells and isolated algal polysaccharide.

Isolation and identification of poplar isoplastocyanins.

An improved four-stage isolation and purification procedure for preparing poplar isoplastocyanins is described in detail. Absorbance (UV-VIS) spectroscopy and isoelectric focusing (IEF) are used to determine the protein purity and identity. The present procedure increases twice the total plastocyanin (PC) yield. Four PC isoform fractions are consecutively isolated at the third chromatographic step: oxidized PCa(II) and PCb(II) and reduced PCb(I) and PCa(I). PCa(II) and PCb(II) obtained at the fourth chromatographic step are highly purified PC isoforms which show the purity index (p.i.) A278/A597 ≤ 0.85. Isoelectric points (pI values) of the PC isoforms are found to be at pH 3.92 ± 0.04 for PCa and at pH 3.85 ± 0.02 for PCb. The results of appropriate biological experiments that include the highly purified poplar PC isoforms could give answers to the questions about the physiological significance of PC dimorphism for photosynthesis.