The effect of sulfite on the ATP hydrolysis and synthesis activities in chloroplasts and cyanobacterial membrane vesicles can be explained by competition with phosphate.

The effect of sulfite on ATP synthesis and hydrolysis activities is investigated in spinach chloroplasts and in membrane vesicles from the cyanobacterium Synechococcus 6716. Sulfite inhibits phenazine methosulfate-mediated cyclic photophosphorylation both in thiol-modulated chloroplasts and in cyanobacterial membranes with HSO3- (bisulfite) as the active ionic species. The observed inhibition is not due to inhibition of electron transfer or to uncoupling by sulfite. ATP synthesis in cyanobacterial membranes is more sensitive to sulfite when the inorganic phosphate concentration is decreased. This indicates competition between sulfite and phosphate for the same binding site on the ATP synthase. In cyanobacterial membranes sulfite can replace a proton gradient as activator of ATP hydrolysis in the same way as in reduced chloroplasts. By modeling, competition between sulfite and phosphate can fully explain the findings concerning both inhibition and activation.

The effect of sulfite on the ATP hydrolysis and synthesis activity of membrane-bound H(+)-ATP synthase from various species.

The action of sulfite on ATP hydrolysis and synthesis activities is investigated in membrane vesicles prepared from the cyanobacterium Synechococcus 6716, chromatophores from the photosynthetic purple bacterium Rhodospirillum rubrum, membrane vesicles from the related non-photosynthetic bacterium Paracoccus denitrificans, and bovine heart submitochondrial particles. Without any further pretreatment ATP hydrolysis is stimulated by sulfite in all four membrane preparations. Typically ATP synthesis in the cyanobacterial membrane vesicles is inhibited by sulfite, whereas ATP synthesis in chromatophores and the submitochondrial particles is not. These differences in sensitivity of ATP synthesis to sulfite, however, correspond well with the distribution of (photosynthetic) sulfur oxidizing pathways in the remaining three organisms/organelles compared in this study.

Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism.

Quinoprotein (2,7,9-tricarboxy-1H-pyrrolo-[2,3-f]quinoline-4,5-dione quinone form (PQQ)-containing) ethanol dehydrogenase (EDH) from Pseudomonas aeruginosa ATCC 17933 was purified to homogeneity. EDH has an alpha 2 beta 2 configuration and subunits comparable in size to those of methanol dehydrogenase (MDH). Compared with other PQQ-containing dehydrogenases, Ca2+ is rather loosely bound and it seems necessary for PQQ binding and stability of EDH. Two soluble cytochromes c were detected in extracts from ethanol-grown cells and both were purified. One of these has an alpha-band at 551 nm for its reduced form, the oxidized form being an excellent electron acceptor for the semiquinone form of EDH. Since this cytochrome is quite different from the already known cytochrome c551 (operating in nitrate respiration) of this organism, it is indicated here as cytochrome cEDH. Comparison of the N-terminal amino acid sequence of cytochrome cEDH with the complete sequence of cytochrome cL (the electron acceptor of MDH), cytochrome cH (the electron acceptor of cytochrome cL) and cytochrome c551 revealed some similarity only to internal stretches of amino acids of the last two. The other soluble cytochrome appeared to be the already-known cytochrome c556. Since it was not an electron acceptor for cytochrome cEDH (neither for EDH), cytochrome cH is lacking in the quinoprotein-EDH-ethanol oxidation system of P. aeruginosa. It seems, therefore, that the respiratory chains for MDH and EDH are different.

Isolation and characterization of cytochrome c550 from the methylamine-oxidizing electron-transport chain of Thiobacillus versutus.

The isolation and purification of cytochrome c550 from the methylamine-oxidizing electron-transport chain in Thiobacillus versutus is reported. The cytochrome is a single-heme-containing type I cytochrome c with a relative molecular mass of 16 +/- 1 kDa, an isoelectric point of 4.6 +/- 0.1, a midpoint potential of 272 +/- 3 mV at pH less than 4 and 255 +/- 5 mV at pH = 7.0, and an axial coordination of the Fe by a methionine and a histidine. The midpoint potential decreases with increasing pH due to the deprotonation of a group tentatively identified as a propionate (pKa = 6.5 +/- 0.1 and 6.7 +/- 0.1 in the oxidized and reduced protein, respectively) and a change in the Fe coordination at pH greater than 10. The electron-self-exchange rate appears to depend strongly on the ionic strength of the solution and is relatively insensitive to changes in pH. At 313 K and pH 5.2 the electron-exchange rate amounts to 0.7 x 10(2) M-1 s-1 and 5.3 x 10(2) M-1 s-1 at I = 40 mM and I = 200 mM, respectively. Amino acid composition and molar absorption coefficients at various wavelengths are reported. Resonances of heme protons and the epsilon H3 group of the ligand methionine of the Fe have been identified in the 1H-NMR spectrum of the reduced as well as the oxidized cytochrome.

Cytochrome b-562 from Acinetobacter calcoaceticus L.M.D. 79.41. Its characteristics and role as electron acceptor for quinoprotein glucose dehydrogenase.

A soluble cytochrome b was purified from Acinetobacter calcoaceticus L.M.D. 79.41. On the basis of the alpha-band maximum of a reduced preparation, measured at 25 degrees C, it is designated as cytochrome b-562. This cytochrome is a basic monomeric protein (pI 10.2; Mr 18,000), containing one protohaem group per molecule. The reduced form, at 25 degrees C, showed absorption bands at 428, 532 and 562 nm. At 77 K the alpha-band shifted to 560 nm (with a shoulder at 558 nm). The reduced cytochrome did not react with CO. Cytochrome b-562 is most probably (loosely) attached to the outside of the cytoplasmic membrane, since substantial amounts of it, equimolar to quinoprotein glucose dehydrogenase (GDH), were present in the culture medium when cells were grown in the presence of low concentrations of Triton X-100. The midpoint potential at pH 7.0 was found to be +170 mV, a value that was lowered to +145 mV by the presence of GDH. Since the GDH was shown to have a midpoint potential of +50 mV, cytochrome b-562 could function as the natural primary electron acceptor. Arguments to substantiate this view and to propose a role of ubiquinone-9 as electron acceptor for cytochrome b-562 are presented.

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis.

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between two HQNO inhibition sites, or more probably in a Q- or b-cycle.

Electron transport and cytochromes in aerobically grown Proteus mirabilis.

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis.

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively. In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).

Localization of glycosidases in the wall of living cells from cultured Convolvulus arvensis tissue.

Nine glycosidase activities were detected in isolated cell walls of cultured Convolvulus arvensis L. cells. Seven were also found in the cytoplasmic fraction. Optimal pH values were all in the acid region. The in vivo localization of some of these glycosidases was studied by assaying whole cells. Suspended cells hydrolysed the externally supplied substrates for α-galactosidase (EC 3.2.1.22), ß-galactosidase (EC 3.2.1.23), ß-glucosidase (EC 3.2.1.21), α-mannosidase (EC 3.2.1.24) and ß-xylosidase (EC 3.2.1.37). Since intracellular enzymes were latent or showed little leakage, the cells were regarded to be relatively intact. In the cases investigated, the apparent glycosidase activity values of whole cells corresponded to those of isolated cell walls. The pH-activity profiles were similar. The Km values were identical indicating equal accessibility to substrate. The enzymes could be partially removed from the cells by strong salt solutions. The data are consistent with an in vivo cell wall localization of a number of glycosidases.