PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe²⁺ or Mg²⁺ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.     

Semiaerobic induction of bacteriochlorophyll synthesis in the green bacterium Chloroflexus aurantiacus.

Comparison of Chloroflexus aurantiacus J-10-fl cells by freeze-fracture electron microscopy showed that cell shape and dimensions did not depend on oxygen tension or light intensity during growth. The major morphological difference between cells cultured anaerobically in the light and aerobically in the dark was the absence of chlorosomes in aerobically grown cells. C. aurantiacus cells cultured aerobically in the dark began bacteriochlorophyll synthesis immediately when shifted to either phototrophic or semiaerobic conditions. Cells adapting to phototrophic conditions grew to the same density and synthesized as much bacteriochlorophyll as nonadapting phototrophic cultures grown at the same light intensity. Cells adapting to reduced oxygen tension (semiaerobic conditions) in the dark entered an 8- to 12-h growth lag during which the bacteriochlorophyll content increased significantly. Despite variations in the initial bacteriochlorophyll content and in the length of the growth lag, the amounts of bacteriochlorophyll a and c were constant at the end of the semiaerobic growth lag. At later times during adaptation to semiaerobic conditions, after growth resumed, variations in the ratio of bacteriochlorophyll c/bacteriochlorophyll a were observed and suggested independent regulation of the two bacteriochlorophylls.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

The adjustment of photosynthetically grown cells of Rhodospirillum rubrum to aerobic light conditions

Summary The growth of photosynthetically precultured cells of Rhodospirillum rubrum under aerobic condition in light is investigated. Special emphasis is given to the question of whether the photosynthetic electron transport chain is influenced under these conditions. Light-induced absorbance changes under anaerobic conditions show that although in whole cells a variation can be noted, the reactions of isolated membranes decrease only very slowly and parallel to each other. The photophosphorylation activity remains constant on a bacteriochlorophyll basis. On a cell mass basis this activity decreases parallel to the decreasing bacteriochlorophyll content. Light-dependent NAD⁺ reduction by ascorbate-DCPI remains constant on a bacteriochlorophyll basis, whereas succinate supported NAD⁺ reduction in light increases. On a cell mass basis the activity of succinate supported NAD⁺ reduction stays nearly constant, thus showing similar responses to the presence of oxygen in light as the NADH oxidase system. NADH oxidase activity increases on a bacteriochlorophyll basis and does not change on a cell mass basis. Parallel to the NADH-oxidase system, oxygen uptake in the dark by whole cells does not change after aerobiosis in light. Light inhibits respiration even after several generations of growth in the presence of oxygen; however, the inhibition decreases slowly. Light inhibition of respiration can be totally overcome by the addition of the uncoupler CCCP. These results indicate that light-dependent electron transport is not influenced significantly by the presence of oxygen. Although the respiratory system is formed, cells preferentially grow photosynthetically. Respiration takes over when the amount of bacteriochlorophyll reaches very low values.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - BChl bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DCPI Na-2,6-dichlorophenol-indophenol - NAD(P)⁺ nictotinamide-adenine-di; nucleotide (phosphate) - NADPH reduced NAD(P)⁺ - TMPD N,N,N-N-tetra; methyl-p-phenyldiamine     

The dependency of proton extrusion in the light on the developmental stage of the photosynthetic apparatus in Rhodospirillum rubrum

The rate of proton extrusion by whole cells of Rhodospirillum rubrum is constant on a bacteriochlorophyll basis only above cellular bacteriochlorophyll concentrations of about 10 nmol bacteriochlorophyll per mg cell protein. At specific bacteriochlorophyll cellular levels below this value, the rate of proton extrusion per bacteriochlorophyll increases. Correspondingly, membrane preparations isolated from these cells exhibit increases in the rate of proton uptake on a pigment basis. Concomitant with variations in the rates of proton extrusion by whole cells, light energy fluxes for saturating this process also vary. A fair proportionality between maximum rates of proton extrusion of whole cells and the bacteriochlorophyll cellular levels above 10 nmol per mg protein indicates that the degree of continuity of intracytoplasmic membranes and of the cytoplasmic membrane remains largely constant.     

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.Abbreviations ALA 5-aminolevulinic acid - R Rhodospirillum - Rsp. Rhodopseudomonas     

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     

Control of composition and activity of the photosynthetic apparatus of Rhodopseudomonas capsulata grown in ammonium-limited continuous culture

Rhodopseudomonas capsulata was grown either phototropically in the light or chemotrophically in the dark at oxygen tensions of 5 mm and 3 mm Hg in ammonium-limited continuous culture. During growth limitation bacteriochlorophyll content of cells and membranes varied dependent on growth rate drastically in chemotrophic cultures. Concomittantly, the ratio of membrane protein to total protein varied in the range of 30-41%. This dependence of membrane differentiation on growth rate was less evident in phototrophically grown cells. The incorporation of the bulk of bacteriochlorophyll was shown to be quantitatively correlated to the incorporation of 1-3 low molecular weight proteins with molecular weights in the range of 14 to less than 10 k daltons. Supported by similar findings of other authors it is proposed, that these proteins are to be attributed to the species of antenna bacteriochlorophyll and represent components of the photosynthetic apparatus. With decreasing growth rates the size of the photosynthetic unit with respect to the population of bacteriochlorophyll- and protein molecules was reduced subsequent to a reduction in the rate of incorporation of antenna-bacteriochlorophyll and the low molecular weight proteins, the reaction-center bacteriochlorophyll content of the membranes remaining constant. A parallel decrease in potential phosphorylating capacity was observed. It is concluded, that under these conditions, primary photochemical reactions in the reaction center were not the rate-limiting step in photophosphorylation. The interaction of growth limitation by an anabolic precursor (NH+4) and control of membrane differentiation by light intensity or oxygen tension is discussed.     

The mechanism of the attachment of esterifying alcohol in bacteriochlorophyll a biosynthesis.

The mechanism through which the C-17(3) carboxy group of bacteriochlorophyllide a is esterified to produce bacteriochlorophyll aphytyl of Rhodopseudomonas spheroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum was studied by using 5-aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms. The latter species was prepared by an exchange reaction in which 5-aminolaevulinate hydrochloride was heated in H218O in an autoclave. A method for the determination of the 18O content of the C-1 oxygen atoms of 5-aminolaevulinate was developed. As a prelude to the mechanistic work, a systematic study was undertaken to establish the optimal conditions under which a significant proportion of the bacteriochlorophyll a of the two photosynthetic organisms originated from the exogenously added 5-aminolaevulinate. It was found that, when Rps. spheroides and Rsp. rubrum were grown in the presence of about 0.15mM- and 1.2mM-5-aminolaevulinate respectively, 30-40% of their chlorophyll was derived from the added precursor. In these conditions, 5-amino[1,4-18O3]laevulinate was incorporated into bacteriochlorophyll aphytyl and bacteriochlorophyll ageranylgeranyl by the relevant organisms. The samples of chlorophylls were then hydrolysed with alkali to obtain phytol and geranylgeraniol, which were converted into the corresponding trimethylsilyl derivatives and analysed by gas chromatography-mass spectrometry. The data were used to deduce that the alcohols contained 90-95% of the 18O originally present at each of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results it is suggested that the ester bond at C-17(3) is formed, not by a chlorophyllase type of enzymic reaction, but by a process involving the nucleophilic attack by the C-17(3) carboxylate group of the chlorophyllide on the activated form of an isoprenyl alcohol.     

Reversible effect of intensive light on photobiochemical properties of Rhodospirillum rubrum chromatophores

The effect of high intensity (photosynthesis-saturating) light on the optical properties of the bacteriochlorophyll and the light-induced H+ uptake by R. rubrum chromatophores was studied. It was shown that under aerobic conditions illumination causes reversible inhibition (in the dark) of the chromatophore ability for the light-induced uptake of H+, a reversible inhibition of the photosynthetical reaction center function and irreversible bleaching of the antennal bacteriochlorophyll. A kinetic comparison of spectral effects and reversible changes in pH as well as the effects of atmospheric oxygen and exogenous electron donors suggests that inhibition of photoactivity of the chromatophores upon illumination is due to accumulation of oxidized bacteriochlorophyll in the reaction center.     

The mechanism of the C-13(3) esterification step in the biosynthesis of bacteriochlorophyll a

5-Aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms was prepared and incorporated into bacteriochlorophyll aphytyl of Rhodopseudomonas sphaeroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum. The biosynthetic samples of the bacteriochlorophylls were separately processed to obtain their isoprenyl alcohol components from the C-17(3) ester linkages and methanol from the C-13(3) methoxycarbonyl group. Methods were developed for the quantification of the isotopic composition of the various alcohols (methanol, phytol, geranylgeraniol). It was shown that the hydroxyl oxygen atoms of all the three alcohols originated from one of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results the in vivo mechanism for the O-methylation reaction at C-13(3) during the biosynthesis of the two species of bacteriochlorophylls is discussed.     

Slow reversible bleaching and fluorescence quenching caused by carotenoid absorption in chromatophores and cells of Chromatium vinosum

Light absorbed by carotenoids in Chromatium can result in photobleaching of bacteriocholorophyll and quenching of B 890 fluorescence, whereas light of the same intensity absorbed by bacteriochlorophyll has no such effect. Photobleching and fluorescence quenching are partly and slowly reversible in the dark. They are prevented by removal of oxygen or by addition of various reductants and decreased by addition of NaN₃₋ histidine or tryptophane. This suggests the participants of singlet excited oxygen in the measured phenomena. As change in temperature between 30° and –30°C and addition of gramicidin S, which changes the permeability of the membranes, does not affect photobleaching or fluorescence quenching markedly, enzymatic or structural properties do not seem to be involved. The results suggest that light absorbed by carotenoids is partly transferred to bacteriochlorophyll and partly used to excite carotenoids to triplet states. The latter process will counteract the function that carotenoids have in protecting chromatophore components against photobleaching.     

Control of bacteriochlorophyll accumulation by light in Rhodobacter capsulatus.

Oxygen levels which control induction of the assembly of the pigment-protein photosynthetic polypeptides in dark-grown Chloroflexus aurantiacus were determined. The induction signal by low-oxygen tension is not directly related to the respiratory competence of these photosynthetic cells. Cytochrome c554, the primary electron donor to P865+ of the reaction center, is not present in dark-grown respiratory cells but is induced in parallel with bacteriochlorophylls a and c and at similar oxygen partial pressure. The development of these components of the photosynthetic apparatus and its electron transport chain is completely independent of the presence of any detectable light or bacteriochlorophyll c or a pigments in C. aurantiacus.     

Control of the formation of bacteriochlorophyll,and B 875- and B 850-bacteriochlorophyll complexes in Rhodopseudomonas sphaeroides mutant strain H5

Rhodopseudomonas sphaeroides mutant H5 lacking 5-aminolevulinic acid synthase was employed to study the control of the formation of total bacteriochlorophyll as well as of the B875- and B850-bacteriochlorophyll protein complexes. The organisms were grown phototrophically in a chemostat where cell protein formation was limited by iron ions and bacteriochlorophyll by 5-aminolevulinic acid. 0.07 mol of bacteriochlorophyll was formed per mol of 5-amino-levulinic acid consumed. This stoichiometric relationship was not influenced by a twelve-fold variation in light energy flux. However, cell protein levels increased and, consequently, cellular specific bacteriochlorophyll contents decreased with increases in light energy flux. The ratio of B875- to B850-pigment protein complexes was inversely proportional to the velocity of 5-aminolevulinic acid supply (mol per cell protein and time) which in this system equals the velocity of 5-aminolevulinic acid consumption and the velocity of bacteriochlorophyll formation. Light had no direct effect on the ratio of B875- per B850-pigment complexes but an indirect effect via its control of protein formation. Changes in the ratio of the two pigment complexes resulted from the fact that significantly lower amounts of 5-aminolevulinic acid supplied per protein and time were required to saturate the system assembling the B875-complexes than that assembling the B850-complexes. The data suggest lack of light-dependent control in the formation of bacteriochlorophyll and its complexes subsequent to the 5-aminolevulinic acid pool.     

Influence of blue light on the structure stability of antenna complexes from Allochromatium minutissimum with different content of carotenoids

Effects of photooxidation of bacteriochlorophyll (absorbtion at 850 nm) from the light-harvesting complex LH2 of Alc. minutissimum membranes on the LH2 complex structure have been studied. Photooxidation was induced by blue light that is absorbed by carotenoids. Four samples with different levels (from 100% to 3–5%) and composition of carotenoids were obtained by inhibiting the carotenoid biosynthesis in bacteria with diphenylamine. Electrophoresis in polyacrylamide gel showed that after illumination LH2 complex contained all the oxidized bacteriochlorophyll. The carotenoid composition did not change after the oxidation of the main part of bacteriochlorophyll in the LH2 complex. The results suggest that oxidation takes place in the bacteriochlorophyll part, which is essential for the molecule optical properties (the system of double conjugated bonds is changed), but does not influence the stability of the structure of the LH2 complex.     

Growth and adaptation to phototrophic conditions of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides at different temperatures

The influence of temperature on yields of cell protein and bacteriochlorophyll as well as on the rates of growth and bacteriochlorophyll synthesis was studied with Rhodospirillum rubrum and Rhodopseudomonas sphaeroides. Under chemotrophic conditions net cell-protein production increased in cultures of both species along with temperature from 14°C up to the optimum at 33°C. Under phototrophic conditions cell-protein yields were largely constant within the range from 21°C to 33°C. At temperatures below 21°C and above 33°C yields decreased. These results are interpreted in terms of coupling between energy yielding or redox equivalent providing metabolisms and cell biosynthesis. Upon adaptation from chemotrophic to phototrophic conditions a direct relationship between temperature increase and bacteriochlorophyll level was observed. Arrhenius plots of both, specific growth rates and rates of bacteriochlorophyll synthesis, revealed discontinuities at about 20°C. Temperature coefficients either above or below those discontinuities were similar in both species. In R. rubrum temperature coefficients of the synthesis of total bacteriochlorophyll were also representative of the synthesis of photochemical reaction center and light harvesting bacteriochlorophylls. But in R. sphaeroides significant differences were observed between temperature coefficients of the syntheses of bacteriochlorophylls of the costantly composed reaction centerlight harvesting complex on one hand and of both, total and the quantitatively variable light harvesting bacteriochlorophylls on the other. The results are interpreted in light of hypotheses on the regulation (a) of cellular bacteriochlorophyll levels as well as (b) of the ratio of functionally different bacteriochlorophylls in the photosynthetic apparatus.Abbreviation Bchl bacteriochlorophyll     

Effect of Oxygen and Light on the Accumulation of Bacteriochlorophyll in the Rhizobial Strain BTAi 1

Strain BTAi 1 is a facultative aerobe and produces maximum amountsof bacteriochlorophyll (BChl) at atmospheric oxygen concentrations.Levels of BChl are reduced by either lowering or raising theambient oxygen concentration. BTAi 1 requires a photoperiodfor maximal accumulation of BChl. The fluence that producesthis maximal accumulation is approximately 35 µmol s^–1m^–2. BChl content is reduced by either lowering or raisingthe fluence from the above concentration. Accumulation of BChloccurs predominantly in the dark, provided that there has beenexposure to a threshold level of light. After an initial lighttreatment, BChl accumulation is detectable after thirty minutesin the dark and continues to increase over the next twenty fourhours. While BChl accumulation is light-stimulated, it is alsolight-inhibited during successive light-dark exposures. In addition,no BChl accumulates in the light after an initial continuousdark treatment, and a prolonged continuous light treatment producesreduced levels of BChl during the dark period. The inhibitionby light is the same under ambient or subambient oxygen concentrations,suggesting that the cause of the inhibition is not simply photodegradationin the presence of oxygen and light. (Received July 21, 1994; Accepted November 16, 1994)     

Oxygen does not directly regulate carotenoid biosynthesis in Rhodopseudomonas capsulata.

We examined the role of bacteriochlorophyll synthesis on the regulation of carotenoid synthesis in Rhodopseudomonas capsulata. Strains capable of making bacteriochlorophyll accumulated greater amounts of carotenoids under low oxygen than they did under high oxygen. However, strains unable to produce bacteriochlorophyll did not regulate their carotenoid production in response to changes in oxygen tension. This indicates that oxygen does not directly regulate carotenoid production.