Four sources of carbonic anhydrase (CA) activity in submembrane preparations of photosystem II (PS II) isolated from pea leaves were examined. Three of them belong to the hydrophilic proteins of the oxygen-evolving complex of PS II with molecular mass 33 kDa (protein PsbO), 24 kDa (protein PsbP), and 18 kDa (protein PsbQ). The fourth source of CA activity is associated with a pigment-protein complex of PS II after removing three hydrophilic proteins by salt treatment. Except for protein PsbQ, the CA activity of all these proteins depends on the presence of Mn2+: the purified protein PsbO did not show CA activity before adding Mn2+ into the medium (concentration of Mn2+ required for 50% effect, EC(50), was 670 microM); CA activity of protein mixture composed of PsbP and PsbQ increased more than 5-fold upon adding Mn2+ (EC(50) was 45 microM). CA activity of purified protein PsbP increased 2-fold in the presence of 200 microM Mn2+. As indicated for the mixture of two proteins (PsbP and PsbQ), Mg2+, Ca2+, and Zn2+, in contrast to Mn2+, suppressed CA activity (both initial and Mn2+-induced activity). Since the found sources of CA activity demonstrated properties different from ones of typical CA (need for Mn2+, insensitivity or low sensitivity to acetazolamide or ethoxyzolamide) and such CA activity was found only among PS II proteins, we cannot exclude that they belong to the type of Mn-dependent CA associated with PS II.

A protective effect of bicarbonate (BC) against extraction of the extrinsic proteins, predominantly the Mn-stabilizing protein (PsbO protein), during treatment of Photosystem II (PS II) membrane fragment from pea with 2 M urea, and at low pH (using incubation in 0.2 M glycine-HCl buffer, pH 3.5 or 0.5 M citrate buffer, pH 4.0-4.5) was detected. It was shown that the extraction of the proteins with Mw 24 kDa (PsbP protein) and 18 kDa (PsbQ protein) by the use of highly concentrated solutions of NaCl does not depend on the presence of BC in the medium. An optimal concentration of BC at which it produces the maximum protecting effect was shown to be between 1 mM and 10 mM. The addition of formate did not influence the protein extraction but it reduced the stabilizing effect of BC. Independence of the stabilizing effect on the presence of the functionally active Mn within the water-oxidizing complex indicates that the protecting effect of BC is not related to its interaction with Mn ions. The fact that there is a preferable sensitivity of the PsbO protein to the absence of BC in the medium during all the treatments makes it possible to suggest that either BC interacts directly with the PsbO protein or it binds to some other sites within PS II and this binding facilitates the preservation of the native structure of this protein.

It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b(559) complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO(3)(-), with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe(2+), it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.

No abstract

A protective action of K15 (4-[methoxy-bis(trifluoromethyl)methyl]-2,6-dinitrophenylhydrazone methyl ketone), an inhibitor of electron transport in photosystem 2 (PS 2), against photoinactivation of the PS 2 reaction center (RC) D1/D2/cytochrome b(559) complex, isolated from pea chloroplasts, by red light (0.7 mmol photons/sec per m(2)) has been investigated under aerobic conditions. The inhibitor K15 causing cyclic electron transfer around PS 2 and thus prohibiting stabilization of separated charges has been shown to effectively protect RC both against the loss of photochemical activity (measured as reversible photoinduced absorbance changes related to photoreduction of pheophytin) and aggregation and degradation of the proteins D2 and D1 during photoinactivation. Comparison of the protective action of K15 and of another inhibitor of electron transfer in PS 2, diuron, against light-induced destruction of proteins D1 and D2 shows that diuron stabilizes protein D1 and K15 stabilizes protein D2. The preferential protection of D2 against photoinduced destruction revealed in our work is in accord with the concept of a specific binding of K15 with this protein. It is proposed that this binding site may be that of the primary quinone electron acceptor Q(A) located on the D2 protein (in contrast to diuron, which is known to replace the secondary electron acceptor Q(B) from its binding site on D1).