Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides.

Cells of Rhodopseudomonas spheroides, strains R-26 or GVP, were grown photosynthetically, disrupted and two particulate fractions separated by sucrose-density-gradient centrifugation. The upper particulate fraction, enriched in bacteriochlorophyll, was identified as containing the chromatophores; the lower particulate fraction had the characteristics of the cell envelope. The two fractions differed in cytochrome content and cytochrome spectra. Ferrochelatase was found almost exclusively in the chromatophore fraction and was located on the outer face of the chromatophores, i.e. in contact with the cytosol in intact cells. The addition of 59FeCl3 to cells growing in low-iron media resulted in labelling of the protohaem fraction (probably arising from cytochrome b) of the membranes. The specific radioactivity of the haem of the chromatophores rose more rapidly than that of the envelope fraction and then after 2 h declined to approximately the same value, suggesting that haems of the chromatophore may act as precursors of haem of the envelope.     

Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected.

With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction.

Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction.

     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

A new membrane-bound b-type cytochrome, cytochrome b-558, from photosynthetically grown Rhodopseudomonas sphaeroides

A new membrane-bound b-type cytochrome, cytochrome b-558, was removed from chromatophore membranes of photosynthetically grown Rhodopseudomonas sphaeroides strain R-26 by deoxycholate-cholate extraction. The cytochrome was purified by ammonium sulfate fractionation and ion-exchange chromatography. Cytochrome b-558 had absorption maxima at 280 and 405 nm in the oxidized form, and at 558, 528, and 420 nm in the reduced form. It had a midpoint potential of--130 mV at pH 7.0. The minimal molecular weight of this protein was 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it contained one mole heme per mole of protein. The isoelectric point was 8.5. The electrophoretic pattern of heme-carrying proteins and the redox potentiometry showed that cytochrome b-558 was present in membranes from wild type, strain R-26, and strain GA grown photosynthetically, but not from any strain grown aerobically.     

THE PREPARATION AND PROPERTIES OF BACTERIAL CHROMATOPHORE FRACTIONS

Chromatophore material from the bacterium Rhodopseudomonas spheroides was freed of ribosomes by centrifugation in 27 per cent RbCl and then separated into "heavy" and "light" fractions by centrifugation through a sucrose gradient. The fractions differed from one another in the following ways. (a) The isopycnic density of the heavy fraction was between 1.15 and 1.18 gm/ml and that of the light fraction was 1.14 gm/ml. (b) The heavy fraction was able to bind ribosomes; the light fraction was not. (c) The light fraction was homogeneous in the ultracentrifuge and had a sedimentation constant, extrapolated to infinite dilution, of 153 s_20,w. The heavy fraction was grossly heterogeneous. (d) Both the amount of bacteriochlorophyll relative to protein and the ratio of bacteriochlorophyll to carotenoids were greater in the light fraction. (e) The spectra of the two fractions in the near infra-red were different. Comparisons of the chromatophore fractions from cells with different amounts of bacteriochlorophyll showed that the specific bacteriochlorophyll contents of the two fractions did not change to the same extent as did that of the whole cells. The amount of heavy fraction from pigmented cells was roughly independent of the cellular pigment content and was about equal to that from pigment-free cells. The amount of light fraction depended on the pigment content of the cells; no light fraction was obtained from cells devoid of bacteriochlorophyll. The cytochrome complements of both fractions underwent quantitative as well as qualitative changes with varying growth conditions. The size of the photosynthetic unit in R. spheroides appeared to increase as the total cellular bacteriochlorophyll content increased; however, the number of units per light fraction particle remained constant.     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Distribution of EIIFru over the membranes of phototrophically grown Rps. sphaeroides

The distribution of the fructose carrier over the membranes of Rhodopseudomonas sphaeroides was studied in cells grown under light saturation and light limitation. Three types of membranes were isolated after disruption of the cells in a French press. All three types were present in the cells grown either under the high or low light intensity, but they were present in different quantities. The cytoplasmic membrane could be separated from the photosynthetic membranes by Sephacryl S-1000 chromatography. The cytoplasmic membrane has the highest specific density and fructose carrier content and does not contain the light-harvesting pigments. The photosynthetic membranes could be resolved into two types by sucrose density gradient centrifugation. Type A predominates when cells are grown under light saturation, whereas type B, the chromatophores, is synthesized abundantly under light limitation. The properties of type A are in between the properties of the cytoplasmic membrane and the chromatophores. It has a slightly lower specific density and contains four times less fructose carrier than the cytoplasmic membrane, but contains half of the light-harvesting bacteriochlorophyll of the chromatophore membrane. The fructose carrier content in the type B membranes, the chromatophores, is very low.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

The electron transport chain of Bacterionema matruchotii

The membrane fraction of Bacterionema matruchotii contains an electron transport chain with oxidizing activity for NADH and succinate. Respiration was inhibited by KCN, 2-heptyl-4-hydroxyquinoline-N-oxide, UV light irradiation and CO. UV light irradiation, analysis of membrane extracts, and reconstitution of respiration in UV light treated membranes suggested that respiration is mediated by a menaquinone derivative. The membranes contained cytochromes a, b, and c. Inhibition studies and the effect of KCN and CO on the cytochrome spectrum indicated the presence of an a+a3 cytochrome oxidase and cytochrome o. The membrane fraction from cells grown under O2-limiting conditions contained nitrate reductase activity. In B. matruchotii, electron transport is coupled to oxidative phosphorylation as judged by the effects of substrates and inhibitors on the intracellular ATP concentration.     

Characterization of the c-type cytochromes of Nitrosomonas europaea with the aid of fluorescent gels

When a total soluble extract of Nitrosomonas europaea was denatured with dodecyl sulphate, subjected to dodecyl sulphate/polyacrylamide-gel electrophoresis and illuminated with near-u.v. light, eight bands of protein fluorescence were observed. All but one of these bands were red in colour, a property characteristic of c-type cytochromes. Standard techniques were used to purify soluble c-type cytochromes from this organism, and it was then possible to assign all but two very minor bands to specific c-type cytochromes, namely hydroxylamine oxidase, cytochrome c-554, cytochrome c-552 and a cytochrome c-550 not previously described. The eight band had fluorescence peaking in the green region of the spectrum, probably caused by covalently bound flavin, and co-purified with hydroxylamine oxidase. The following physical properties were determined for these components: isoelectric point, molecular weights according to gel filtration and mobility on dodecyl sulphate/polyacrylamide gels, and alpha-band spectra at room temperature and 77K. Redox potentials were measured as follows: cytochrome c-554, E(m,7) = +20mV; cytochrome c-552, E(m,7) = +230mV; cytochrome c-550, E(m,7) = +140mV. When washed membranes were applied to dodecyl sulphate/polyacrylamide gels in the same way, a number of fluorescent bands were observed that could be matched by soluble proteins. In addition, there was one band that could not be detected in supernatants, migrating with an apparent molecular weight of 24000. This species is probably coincident with a c-type cytochrome having E(m,7) = +170mV found in redox titration of these membranes. In future studies, gel fluorescence should form a useful complement to spectroscopy for analysis of cytochrome composition in active cell-free preparations or semi-purified material.     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

Rhodobacter sphaeroides spd mutations allow cytochrome c2-independent photosynthetic growth.

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis.     

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).     

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.     

Photopigments in Rhodopseudomonas capsulata cells grown anaerobically in darkness.

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Cytochemical studies of mitochondria. II. Enzymes associated with a mitochondrial membrane fraction

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained approximately 12 per cent of the protein N and approximately 35 per cent of the phospholipide of the whole mitochondria and accounted for approximately 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

Kinetic and thermodynamic properties of membrane-bound cytochromes of aerobically and photosynthetically grown Rhodopseudomonas spheroides

A green mutant of Rhodopseudomonas spheroides was isolated in which spectroscopic measurements of the α-band region of cytochromes could be made. It was grown either aerobically or photosynthetically, and the membrane fractions prepared from cells of each type. Anaerobic potentiometric titration at 560 nm minus 542 nm showed the same three redox components, tentatively identified as b-type cytochromes, in membrane fractions from either type of cell. The mid-point potentials were approximately +185, +41 and ?104 mV. In membranes from photosynthetically grown cells the major cytochrome form absorbing at 560 nm had a mid-point potential of +42 mV; in aerobically grown cells the major form had a potential of +185 mV. In both types of cell only one c-type cytochrome was found, with a mid-point potential of +295 mV. An a-type cytochrome was present only in aerobically-grown cells.Substrate-reduced particles from these cells were mixed with air-saturated buffer in a stopped-flow spectrophotometer and the kinetics of oxidation of b- and c-type cytochromes were measured. The same two b-type components, reacting with pseudo first order kinetics, were detected in particles from both aerobically and photosynthetically grown cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for oxidation 1.3 s and 0.13 s). The c-type cytochrome of particles from aerobically grown cells was oxidised with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.97 s; the c-type cytochrome of photosynthetic cells was oxidised faster, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.27 s.These observations have implications on the adaptive formation of electron transport systems that are discussed.     

Early formation of intracytoplasmic membranes in Rhodospirillum rubrum

Cytoplasmic and intracytoplasmic membranes were isolated from Rhodospirillum rubrum by equilibrium sucrose density gradient centrifugation. Immediately after the induction of photosynthetically active intracytoplasmic membranes, bacteriochlorophyll is incorporated predominantly into the cytoplasmic membrane. With increasing pigment concentrations the newly arising intracytoplasmic membranes become sites of preferential bacteriochlorophyll incorporation. During this process the infrared absorption band of the pigment shows a red shift. The shift is more pronounced with intracytoplasmic than with cytoplasmic membranes. Pulse-chase of cytoplasmic membrane proteins reveals that such proteins become constituents of intracytoplasmic membranes.     

Membranes of Rhodopseudomonas spheroides: Interactions of Chromatophores with the Cell Envelope

Under carefully controlled ionic conditions, large-scale preparations of highly purified chromatophores and cell envelopes were obtained from phototrophically grown Rhodopseudomonas spheroides by zonal ultracentrifugation. The majority of the bacteriochlorophyll a was located in a single, discrete chromatophore band, whereas the envelopes were nearly devoid of photopigment. The envelope fraction contained substantial quantities of succinic dehydrogenase and cytochromes, confirming that phototrophically grown cells contain a photopigment-deficient cytoplasmic membrane. Magnesium at concentrations of 1.0 mM or higher caused chromatophores to reversibly aggregate with the cell envelope. Significant aggregation was also promoted by other divalent metals (Co(2+) > Mn(2+) > Ca(2+) > Mg(2+)), but aggregation was less extensive with monovalent cations. These results account for the distribution of photopigments in two bands reported by others and further suggest that the photosynthetic apparatus of R. spheroides is located on membranes largely distinct from the cell wall-cytoplasmic membrane complex.     

Control of δ-aminolaevulate synthetase activity in Rhodopseudomonas spheroides

1. delta-Aminolaevulate synthetase from Rhodopseudomonas spheroides grown semi-anaerobically undergoes a spontaneous activation during the first hour after the disruption of cells when homogenates are stored at 4 degrees . 2. After cultures of R. spheroides growing semi-anaerobically are oxygenated no activation of delta-aminolaevulate synthetase occurs in cell extracts. Cessation of activation in extracts is almost complete 10min. after oxygenation of cells has begun. 3. A heat-stable fraction of low molecular weight from semi-anaerobic cells reactivates delta-aminolaevulate synthetase in extracts of oxygenated cells and appears to contain a compound responsible for the spontaneous activation. 4. A heat-stable fraction of low molecular weight from oxygenated cells inhibits the spontaneous activation in extracts of semi-anaerobic cells. 5. The effect of oxygen on the rate of bacteriochlorophyll synthesis in R. spheroides may be mediated through alterations in the concentrations of a low-molecular-weight activator and inhibitor of delta-aminolaevulate synthetase.