Tatiana Shutova

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus ☆

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp(241) is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.

A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3-5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD). Significance and possible functions of CA for the PS II donor side are discussed.

The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii

Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

Time-Resolved Delayed Chlorophyll Fluorescence to Study the Influence of Bicarbonate on a Green Algae Mutant Photosystem II

Investigations On Photosystem II (Psii) Electron Transfer Processes by Observing Transient Absorption Changes Are Challenging. They Require High Psii Concentrations, Extended Averaging and are Negatively Affected By The Strong Light Scattering of Various Sample Types. Avoiding These Problems, The Analysis of the Time-Resolved Delayed Fluorescence (Df) measurements has become an important tool to study quantitatively light-induced electron transfer as well as associated Processes (E.G. Proton Movements) at the Donor Side Of Psii. Inter Alia This method can provide insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The delayed emission of chlorophyll fluorescence was measured on both wild type and mutant of green algae psii membrane particles in the time domain from 10 μs to 60 ms after each flash of a train of nanosecond laser pulses. The influence of the psii-associated carbonic anhydrase (ca) on the donor side reactions was studied for a Chlamydomonas Reinhardtii Wild type and Ca-Free Mutant.

Studies on the structural and functional role of the extrinsic manganese stabilizing protein in higher plants

The PsbO protein acts as a regulatory extrinsic subunit that also stabilizes the manganese cluster of the water oxidizing complex (WOC). This protein often referred to as MSP (manganese stabilizing protein) is present in all oxygen evolving organisms and characterized by striking sequence homologies of the copies from ancient cyanobacteria up to the evolutionary level of higher plants. The overall conformation of the isolated MSP in solution is best described as a thermostable, molten globule state. In order to gather more detailed information on the possible role of the C terminal domain of the MSP comparative studies were performed on the thermal transition of the protein isolated from pea and a synthetic peptide with the C-terminus (KDVKIQGVWYAQLES) sequence of the MSP. Measurements of intrinsic fluorescence emission, circular dichroism in far and near UV and fluorescence polarisation reveal that the cooperativity factor and melting point of MSP depends on pH and the presence of Ca2+ and Mn2+. Furthermore, the reconstitution of oxygen evolution by addition of the solubilized protein to MSP depleted PS II membrane fragment is impaired by the presence of the synthetic peptide thus indicating its interaction with the MSP-binding site of PS II. A possible role of the C-terminus for the structure and function of MSP are discussed.