Calcium binding to the subunit c of E. coli ATP-synthase and possible functional implications in energy coupling.

The 8-kDa subunit c of the E. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mutants, Asp61-->Asn and Asp61-->Gly. The Ca++ binding is pH dependent in both the E. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation. The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.

Induction of an electrogenic transfer of monovalent cations (K+, NH4+) in thylakoid membranes by N,N'-dicyclohexylcarbodiimide.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on photosynthetic electron transport and light-induced NH4+ and K+ uptake in the presence of ammonium or nigericin was studied. DCCD alone had no effect on either the electron transport or the uptake of protons. The simultaneous action of DCCD and low concentrations of ammonium or nigericin was shown to lead to a significant increase in the electron transport rate and a decrease in the steady-state uptake value of H+ and NH4+ or K+. The effect of DCCD on these processes was compared with the effect of the ionophore, valinomycin, which transports potassium and ammonium cations through membranes. The conclusion was made that 1.0-1.5 mol DCCD per mol chlorophyll activated the transfer system of monovalent cations (K+ and NH4+) in thylakoid membranes.

[Correlation between non-cyclic and pseudocyclic electron transport in pea chloroplasts: dependent on the concentration of ferredoxin]. / Sootnoshenie mezhdu netsiklicheskim i psevodotsiklicheskim transportom élektronov v khloroplastakh gorokha v zavisimosti ot kontsentratsii ferredoksina.

The effects of ferredoxin concentrations on the correlation between pseudocyclic and non-cyclic electron transport in isolated pea chloroplasts with NADP+ as an electron acceptor was studied. The correlation of psuedocyclic and non-cyclic electron transport upon photoreduction of NADP+ in vitro can be regulated by ferredoxin concentration. At ferredoxin concentrations studied an addition of NADP+ to the system which makes possible electron transfer from ferredoxin to ferredoxin-NADP+-reductase increases the rate of overall electron transport across the chain. Unlike the pseudocyclic electron transport, photoreduction of NADP+ reaches its maximal saturation at relatively low (10-15 microM) concentrations of ferredoxin (Km = approximately 2-5 microM).

[Electron transport and photophosphorylation, coupled with photoreduction of oxygen by chloroplasts of peas, grown under different conditions of illumination]. / Transport élektronov i fotofosforilirovanie, sviazannye s fotovosstanovleniem kisloroda khloroplastami gorokha, vyrashchennogo pri razlichnoi osveshchennosti.

A comparative study of the intensity of O2 uptake, NADP+ reduction and ATP synthesis in pea chloroplasts grown upon different illumination conditions was carried out. The rates of O2 uptake in the absence of cofactors and in the presence of ferredoxin are almost identical for "dark" and "light" chloroplasts. In a medium containing oxidized NADP+ the rate of electron transport is considerably lower in the "dark" chloroplasts; the pseudocyclic transport coupled with simultaneous reduction of O2 in these chloroplasts is somewhat higher (on the average by 30%). The decrease of electron transfer to NADP+ caused by reduction of light intensity during plant growth (presumably due to the limiting step, i. e. ferredoxin--NADP+-reductase) is to some extent compensated for by a more effective utilization of O2 as an electron acceptor. This eventually results in an increase of the ATP/NADPH ratio.

[Role of ferredoxin in pseudo-cyclic electron transport in isolated pea chloroplasts]. / Uchastie ferredoksina v psevdotsiklicheskom elektronnom transporte v izolirovannykh khloroplastakh gorokha.

The pseudo-cyclic electron transport in isolated pea chloroplasts in a medium without exogenous cofactors but containing methylviologen and ferredoxin was studied. In order to establish the steps limiting the electron transport, the rates of electron transport a different pH values and under phosphorylating and non-phosphorylating conditions in the presence of the uncoupler--gramicidin D--were determined. It was shown that in the absence of exogenous cofactors the total electron transport is limited by a stage of electron transfer from the acceptor site of photosystem I to oxygen. An addition of ferredoxin or methylviologen stimulates oxygen consumption. It was shown that the plot of the dependence of the rate of oxygen consumption on ferredoxin concentration is two-phase in contrast to that of methylviologen concentration, which is indicative of the existence of two competitive reactions of oxygen reduction involving ferredoxin (with different Km values for the protein).

Characterization of plastocyanin from corn (Zea mays L.) and pea (Pisum sativum L.) leaves.

A comparison of plastocyanin isolated from pea and corn leaves was made according to a number of indices. No appreciable differences were detected between the proteins in molecular weight or sedimentation constants. In addition, it was shown that plastocyanin of corn, in comparison with the pea protein, possesses greater thermal stability, is more resistant to the action of high and low H+ ion concentrations, as well as to the action of concentrated solutions of urea and guanidine nitrate. The detected differences indicate peculiarities in the molecular organization of plastocyanins from various groups of plants. It is suggested that the differences noted in the structure of plastocyanins can ensure effective functioning of proteins under nonuniform conditions of the external environment.