Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH

The effects of Cl⁻, Mn²⁺, Ca²⁺, and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl⁻, and strongly inhibited above this concentration. This enzyme is activated by Mn²⁺ and stimulated somewhat by Ca²⁺. The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl⁻ up to 0.4 M. Ca²⁺ appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn₄ complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.     

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a)

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.     

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, Q_A, and the secondary plastoquinone acceptor, Q_B, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced Q_B binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between Q_A and Q_B, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.     

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F_400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q_A⁻ accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q_A⁻ accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t_1/2 ∼ 30 ms. This alternative electron donor is most probably ascorbate.