BBA - Bioenergetics (v.1456, #2-3)

Interactions of arsenate, sulfate and phosphate with yeast mitochondria by Paulina Cortés; Vicente Castrejón; José G Sampedro; Salvador Uribe (67-76).
In the presence of K+, addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (ΔΨ) and this is prevented by millimolar concentrations of phosphate (PO4). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO4) and sulfate (SO4) were as efficient as PO4 to protect mitochondria against the K+ mediated swelling, depletion of the ΔΨ, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO4, SO4 or AsO4 was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO4 and AsO4 on the F1F0-ATPase were tested: both SO4 and AsO4 inhibited the synthesis of ATP following competitive kinetics against PO4 and non-competitive kinetics against ADP. The mersalyl sensitive uptake of 32PO4 was not inhibited by SO4 or AsO4, suggesting that the synthesis of ATP was inhibited at the F1F0-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO4 or sulfite (SO3) were added. It is suggested that the structure and charge similarities of PO4, AsO4 and SO4 result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K+; at the second site, in the F1F0-ATPase, AsO4 and SO4 compete for binding against PO4 leading to inhibition of the synthesis of ATP.
Keywords: Arsenate; ATP synthesis; F1F0-ATPase; Phosphate; Permeability transition pore; Sulfate; Yeast mitochondria;

The H+-ATPase from chloroplasts, CF0F1, was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[α-32P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[α-32P]ADP, the covalently bound label was found exclusively at β-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[α-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 120 s−1, and the rate of ATP hydrolysis was 20 s−1, both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF0F1 and with non-modified CF0F1. The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.
Keywords: H+ATPase; CF0F1; Nucleotide binding; Uni-site catalysis; 2-Azido-nucleotide;

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat by Leslie E. Eisele; Sasha H. Bakhru; Xuemei Liu; Robert MacColl; Mercedes R. Edwards (99-107).
C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57°C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50°C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65°C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the α and β polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.
Keywords: High temperature eukaryote; Phycocyanin; Biliprotein; Cyanidium caldarium;

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15×10−6 Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H2 18O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of 16O2, 16O18O and 18O2 simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S0 and S1 leads to S2 and S3, thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y1 and Y2 are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H2O2 which is decomposed under O2 evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.
Keywords: Cyanobacterium; Photosynthesis; Water splitting; Cooperativity; Mass spectrometry; Oxygen isotope;

Reaction of Escherichia coli cytochrome bo 3 and mitochondrial cytochrome bc 1 with a photoreleasable decylubiquinol by Kirk C Hansen; Brian E Schultz; Guangyang Wang; Sunney I Chan (121-137).
In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3′,5′-bis(carboxymethoxy)benzoin (BCMB) through a carbonate linkage. The resulting compound, DQ-BCMB, was highly soluble in aqueous detergent solution, and showed no reactivity with quinol-oxidizing enzymes prior to photolysis. Upon photolysis in acetonitrile, 5,7-bis(carboxymethoxy)-2-phenylbenzofuran, carbon dioxide, and decylubiquinol were formed. In aqueous media, free 3′,5′-bis(carboxymethoxy)benzoin was also produced. Photolysis of DQ-BCMB with a 308 nm excimer laser led to the release of the BCMB group in less than 10−6 s. Decylubiquinol was released in the form of a carbonate monoester, which decarboxylated with an observed first-order rate constant of 195–990 s−1, depending on the reaction medium. Yields of decylubiquinol as high as 35 μM per laser pulse were attained readily. In the presence of Escherichia coli cytochrome bo 3, photolysis of DQ-BCMB led to the oxidation of quinol by the enzyme with a rate that was limited by the rate of the decylubiquinol release. Mitochondrial cytochrome bc 1 reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b heme reduction and somewhat slower c heme reduction. Oxidation of photoreleased ubiquinol by this enzyme showed saturation kinetics with a K m of 3.6 μM and a k cat of 210 s−1. The saturation behavior was a result of decylubiquinol being released as a carbonate monoester during the photolysis of DQ-BCMB and interacting with cytochrome bc 1 before decarboxylation of this intermediate yielded free decylubiquinol. The reaction of cytochrome bc 1 and photoreleased decylubiquinol in the presence of antimycin A led to monophasic b heme reduction, but also yielded slower quinol oxidation kinetics. The discrimination of kinetic phases in the reaction of cytochrome bc 1 with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cycle in this enzyme.
Keywords: Cytochrome bc 1; Cytochrome bo 3; Photoreleasable substrate; Respiratory chain; Transient absorption spectroscopy; Ubiquinol;