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PubMed1998

CD-monitored redox titration of the Rieske Fe-S protein of Rhodobacter sphaeroides: pH dependence of the midpoint potential in isolated bc1 complex and in membranes.

The redox potential of the Rieske Fe-S protein has been investigated using circular dichroism (CD)-spectroscopy. The CD features characteristic of the purified bc1 complex and membranes of Rhodobacter sphaeroides were found in the region between 450 and 550 nm. The difference between reduced and oxidized CD-spectra shows a negative band at about 500 nm with a half of width 30 nm that corresponds to the specific dichroic absorption of the reduced Rieske protein (Fee, J.A. et al. (1984) J. Biol. Chem. 259, 124-133; Degli Esposti, M. et al. (1987) Biochem. J. 241, 285-290; Rich, P.R. and Wiggins, T.E. (1992) Biochem. Soc. Trans. 20, 241S). It was found that the redox potential at pH 7.0 for the Rieske center in the isolated bc1 complex and in chromatophore membranes from the R-26 strain of Rh. sphaeroides is 300 +/- 5 mV. In chromatophores from the BC17C strain of Rh. sphaeroides, the Em value measured for the Rieske iron-sulfur protein (ISP) was higher (315 +/- 5 mV), but the presence of carotenoids made measurement less accurate. The Em varied with pH in the range above pH 7, and the pH dependence was well fit either by one pK at approximately 7.5 in the range of titration, or by two pK values, pK1 = 7.6 and pK2 = 9.8. Similar titrations and pK values were found for the Rieske Fe-S protein in the isolated bc1 complex and membranes from the R-26 strain of Rb. sphaeroides. The results are discussed in the context of the mechanism of quinol oxidation by the bc1 complex, and the role of the iron sulfur protein in formation of a reaction complex at the Qo-site.

Cell Membrane↗

PubMed2001

Absorption changes induced by the binding of triazines to the QB pocket in reaction centers of Rhodobacter capsulatus.

Inhibitors which block electron transfer from the primary (Q(A)) to the secondary (Q(B)) quinone of the bacterial reaction center are competing with the pool ubiquinones for binding at the Q(B) pocket. Due to the much greater stability of the semiquinone state Q(B)(-) compared with fully oxidized or reduced quinone, a displacement of the inhibitors takes place after one flash from state Q(A)(-)I to state Q(A)Q(B)(-). This process can be monitored from near-IR absorption changes which reflect local absorption shifts specific to Q(A)(-) and Q(B)(-). An anomalous behavior was observed when using triazines in chromatophores of R. capsulatus: the IR absorption change reflecting the formation of Q(B)(-) after one flash was absent. A normal transient decay of this signal was, however, triggered by a second flash, followed by a rapid return to the baseline. We show that this phenomenon is due to an absorption change induced by inhibitor binding (thus present in the dark baseline), with a spectrum close to that of Q(B)(-), so that the Q(B)(-) changes are canceled out during the inhibitor displacement process. On the second flash, one monitors the destruction of the semiquinone, leading transiently to the Q(A)Q(B) state, followed by inhibitor rebinding. This allows a direct measurement of the binding kinetics. This behavior was observed both in chromatophores and in isolated reaction centers from R. capsulatus, but not in R. sphaeroides.

Atrazine↗

PubMed1991

Proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: purification, characterization, and reconstitution.

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4 mumol of PPi hydrolyzed min-1 mg-1 at pH 7.5 and 20 degrees C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56,000. The oligomeric structure of the H(+)-PPi synthase is discussed.

Centrifugation, Density Gradient↗

PubMed1990

Use of the membrane-impermeable guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate to demonstrate the orientation of light-harvesting proteins in Rhodobacter sphaeroides.

The radiolabeled guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate reacts with the epsilon-amino groups of accessible lysyl residues of membrane proteins under relatively mild labeling conditions, yielding labeled homoarginyl residues. Model studies have shown that the resulting homoarginyl residues do act as new cleavage sites for trypsin, but only at a very slow rate of hydrolysis. The reagent has been shown to be impermeable to the intracytoplasmic membranes of Rhodobacter sphaeroides: when cytoplasmic-side-out chromatophores were treated with the reagent, it reacted with all four of the light-harvesting proteins, all of which have one or more lysyl residues on the N-terminal sides of their hydrophobic regions. However, when periplasmic-side-out vesicles, prepared by cytochrome c affinity chromatography, were treated with the guanidinating reagent, three of the light-harvesting proteins (B850 alpha, B850 beta, and B870 beta) were not labeled. The only light-harvesting protein to be labeled (B870 alpha) was the only one of the four to have a lysyl residue on the C-terminal side of its hydrophobic region. Guanidinated B870 alpha polypeptides from both the cytoplasmic-side-out chromatophores and the periplasmic-side-out membrane vesicles were purified and digested with trypsin. The resulting peptide fragments were then separated by high-performance liquid chromatography and analyzed for radioactivity. The results have confirmed the asymmetric orientation of the light-harvesting proteins of R. sphaeroides, with their N-termini on the cytoplasmic side of the intracytoplasmic membrane. In the case of the B870 alpha subunit, the protein has been shown to be transmembrane with its C-terminus on the periplasmic side of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

The extra fragment of the iron-sulfur protein (residues 96-107) of Rhodobacter sphaeroides cytochrome bc1 complex is required for protein stability.

Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc(1) complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPDelta(96-107), ISP(96-107)A, and ISP(104-107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc(1) complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc(1) activity found in the complement chromatophores. The loss (or decrease) of bc(1) activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c(1) into the membrane, the binding affinity of cytochrome b to cytochrome c(1), or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99.

Amino Acid Sequence↗

PubMed1997

Membrane localization, topology, and mutual stabilization of the rnfABC gene products in Rhodobacter capsulatus and implications for a new family of energy-coupling NADH oxidoreductases.

The rnf genes in Rhodobacter capsulatus are unique nitrogen fixation genes that encode potential membrane proteins (RnfA, RnfD, and RnfE) and potential iron-sulfur proteins (RnfB and RnfC). In this study, we first analyzed the localization and topology of the RnfA, RnfB, and RnfC proteins. By activity and immunoblot analysis of expression of translational fusions to Escherichia coli alkaline phosphatase, RnfA protein was shown to span the chromatophore membrane with its odd-numbered hydrophilic regions exposed to periplasm. By alkaline treatment of membrane fractions and following immunoblot analysis using antibodies against recombinant proteins expressed in E. coli, both RnfB and RnfC proteins were revealed to situate at the periphery of the chromatophore membranes. Second, mutual interaction of the Rnf proteins was analyzed by immunochemical determinations of RnfB and RnfC proteins in rnf mutants and their complemented derivatives. The contents in cellular fractions indicated that RnfB and RnfC stabilize each other and that the presence of RnfA is necessary for stable existence of both proteins. These results support a hypothesis that the Rnf products are subunits of a membrane complex. Finally, we detected homologs of rnf genes in Haemophilus influenzae and Vibrio alginolyticus by data base searches and in E. coli by cloning of a fragment of an rnfA homolog followed by a data base search. Close comparisons revealed that RnfC has potential binding sites for NADH and FMN which are similar to those found in proton-translocating NADH:quinone oxidoreductases and that RnfA, RnfD, and RnfE show similarity to subunits of sodium-translocating NADH:quinone oxidoreductases. We predict that the putative Rnf complex represents a novel family of energy-coupling NADH oxidoreductases.

Amino Acid Sequence↗

PubMed2003

Reconstitution of carotenoids into the light-harvesting complex B800-850 of Chromatium minutissimum.

Chromatophores and peripheral light-harvesting complexes B800-850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800-850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.

Bacterial Proteins↗

PubMed2001

Mutations in the gene encoding B, a novel transporter protein, reduce melanin content in medaka.

Pigmentation of the skin is of great social, clinical and cosmetic significance. Several genes that, when mutated, give rise to altered coat color in mice have been identified; their analysis has provided some insight into melanogenesis and human pigmentation diseases. Such analyses do not, however, fully inform on the pigmentation of lower vertebrates because mammals have only one kind of chromatophore, the melanocyte. In contrast, the medaka (a small, freshwater teleost) is a suitable model of the lower vertebrates because it has all kinds of chromatophores. The basic molecular genetics of fish are known and approximately 70 spontaneous pigmentation mutants have been isolated. One of these, an orange-red variant, is a homozygote of a well-known and common allele, b, and has been bred for hundreds of years by the Japanese. Here, we report the first successful positional cloning of a medaka gene (AIM1): one that encodes a transporter that mediates melanin synthesis. The protein is predicted to consist of 12 transmembrane domains and is 55% identical to a human EST of unknown function isolated from melanocytes and melanoma cells. We also isolated a highly homologous gene from the mouse, indicating a conserved function of vertebrate melanogenesis. Intriguingly, these proteins have sequence and structural similarities to plant sucrose transporters, suggesting a relevance of sucrose in melanin synthesis. Analysis of AIM1 orthologs should provide new insights into the regulation of melanogenesis in both teleosts and mammals.

Animals↗

PubMed1965

THE PHOTOASSIMILATION OF SUCCINATE TO HEXOSE BY RHODOSPIRILLUM RUBRUM.

1. A pathway for the synthesis of hexose from succinate by Rhodospirillum rubrum is proposed. 2. With 2,3-(14)C(2)-labelled succinate and fumarate as substrates in experiments with chromatophores and a soluble enzyme fraction of R. rubrum it was found that the products of succinate metabolism by the extracts were the same as in whole cells. It was also found that the light-dependent oxidation of succinate was catalysed by the chromatophores, but that all the other enzymes involved were in the soluble fraction. 3. By using specific assays the presence of all the enzymes required for the proposed pathway was demonstrated in the extracts and their specific activities were measured. 4. The overall rate of succinate assimilation was measured manometrically. The activities of the enzymes assayed were sufficient to account for the overall rate of assimilation. It is concluded that the proposed pathway represents the major mechanism for synthesis of hexose from succinate in R. rubrum. 5. The formation of alanine and aspartate was observed in experiments with isotopically labelled substrates, and possible synthetic pathways for these compounds are discussed.

Adenosine Triphosphate↗

PubMed2000

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.

Antimycin A↗

PubMed1983

Spatial correlation between primary redox components in reaction centers of Rhodopseudomonas sphaeroides measured by two electrical methods in the nanosecond range.

Relative distances between the the primary donor P, the intermediary pheophytin acceptor H, and the iron-quinone acceptor Q of bacterial reaction centers were determined by recording laser flash-induced photovoltages in two experimental systems with nanosecond time resolution. In one system a suspension of chromatophores was subjected to a light gradient and in the other system chromatophores were spread at a heptane/water interface. The 10-ns back reaction occurring in reaction centers with reduced Q could be time resolved. The initial photovoltage amplitude under conditions in which the charge separation proceeded up to the state [P(+)H(-)] was about (2/3) of that when it proceeded up to the state [P(+)HQ(-)]. If the amplitude of the photovoltage is considered to be proportional to the spatial displacement of charges, this result means that pheophytin lies closer to Q than to P.

Journal Article↗

PubMed1995

Identification of amino acid residues involved in structural and ubiquinone-binding functions of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides.

Previous studies established that subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides is involved in structural and ubiquinone-binding functions of the complex. To identify regions or amino acid residues responsible for these functions, deletion, insertion, and substitution mutations at various regions of subunit IV were generated and characterized. Mutational effects on the structural role of subunit IV are indicated by a delay in photosynthetic growth and by a decrease in the cytochrome bc1 complex activity in chromatophores upon detergent treatment. An effect on the ubiquinone-binding function of subunit IV is suggested by an increase in the apparent Km for 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol (Q2H2) of the complex. RSIV delta (2-5), in which residues 2-5 are deleted, had photosynthetic growth behavior, tolerance to detergent treatment, and an apparent Km for Q2H2 of its cytochrome bc1 complex similar to those of wild-type or complement cells, indicating that amino acid residues 2-5 are not essential for subunit IV function. RSIV delta (2-11), with residues 2-11 missing, showed a 24-h delay in photosynthetic growth and a 65% inactivation of the cytochrome bc1 complex upon dodecyl maltoside solubilization. However, its apparent Km for Q2H2 was the same as in wild-type cells, indicating that deletion of amino acid residues 6-11 results in loss of the structural but not the ubiquinone-binding function of subunit IV. RSIV delta (113-124), which has 13 amino acid residues deleted from the C terminus, had photosynthetic growth behavior, tolerance to detergent treatment, and ubiquinone-binding kinetics similar to those of wild-type or complement cells, indicating that residues 113-124 are not essential. Point mutants RSIV(W79L) and RSIV(W79F), in which tryptophan 79 was replaced with leucine or phenylalanine, showed a 24-h delay in photosynthetic growth, a decrease of 75% of the cytochrome bc1 complex activity in chromatophores upon detergent solubilization, and a 4-fold increase in the apparent Km for Q2H2, indicating that Trp-79 is essential for the structural and ubiquinone-binding functions of subunit IV.

Amino Acid Sequence↗

PubMed1966

Photoreduction of Pyridine Nucleotide by Subcellular Preparations from Rhodopseudomonas spheroides.

We have investigated the photoreduction of pyridine nucleotides by crude extracts and chromatophores of Rhodopseudomonas spheroides.Our findings are as follows:NADP is preferentially photoreduced by crude extracts (37,000 x g supernatant fraction) and there is no requirement for the addition of exogenous substrates. Crude extracts also catalyze a nonphotosensitive reduction of NAD.NADP photoreduction is completely inhibited if an NADH trapping system is present and indicates that NADH is required for NADP photoreduction.Washed chromatophores (150,000 x g pellet) do not catalyze NADP photoreduction unless the supernatant fraction is added. The restoring effect of supernatant fraction is lost upon boiling and dialysis. However, supernatant materials can be replaced by an NADH generating system. There is no requirement for anaerobic conditions.Evidence has been presented which suggests that Rhodopseudomonas spheroides contains an energy-linked transhydrogenase that can be driven by a high energy intermediate generated by light or ATP. This intermediate may also be functional in ATP synthesis. The synthesis of ATP and the ATP-supported transhydrogenase is inhibited by oligomycin. This inhibitor does not affect the light-mediated reaction.

Journal Article↗

PubMed1978

Photooxidative Damage in Photosynthetic Activities of Chromatium vinosum.

The capacity of photosynthetic CO(2) fixation in the anaerobic purple-sulfur bacterium, Chromatium vinosum is markedly impaired by strong illumination (9 x 10(4) lux) in the presence of 100% O(2). In the absence of HCO(3) (-), decline in activity occurred gradually, with about 40% of the initial activity remaining after a 1-hour incubation. The addition of 50 millimolar HCO(3) (-) to the incubation medium resulted in a measurable delay (about 30 minutes) of the inactivation process. Ribulose-1,5-bisphosphate carboxylase activity and light-dependent O(2) uptake (electron flow) or crude extracts prepared after pretreatment of the bacterial cells with O(2) and light were not affected but the photophosphorylation capacity of either bacterial cells or chromatophores was drastically reduced. The inhibition of photophos-phorylation in the chromatophore preparations was significantly reduced by the addition of either an O(2) (-) scavenger, Tiron, or an (1)O(2) scavenger, alpha-tocopherol. These results suggest that the active O(2) species, O(2) (-) or (1)O(2), might take part in the observed inactivation.The pretreatment of the bacteria with O(2) and light inhibited CO(2) assimilation through the Calvin-Benson cycle, while relatively stimulating the formation of aspartate and glutamate. It also inhibited the conversion of glycolate to glycine, resulting in a sustained extracellular excretion of glycolate. The inactivation of photosynthetic CO(2) fixation by intact cells was enhanced by low temperature, KCN, or methylviologen addition during the pretreatment with O(2) and light. The mechanism(s) of O(2)-dependent photoinactivation of photosynthetic activities in Chromatium are discussed in relation to the possible role of photorespiration as a means of producing CO(2) in the photosynthetic system.

Journal Article↗

PubMed1981

Antibodies to the F1-ATPase of Rhodospirillum rubrum and its purified native beta-subunit: inhibition of ATP-linked activities in R. rubrum and in lettuce.

1. Antibodies prepared against the Rhodospirillum rubrum F1-ATPase (RrF1) and its purified, native-beta-subunit, exhibited cross-reactivity with the following soluble preparations of R. rubrum ATPase: RrF0 . F1, RrF1 and the beta-subunit. Anti-RrF1, but not anti-beta antibodies, also formed precipitin lines with soluble beta-less Rrf1, indicating that antigenic determinants of both the beta-subunit and the other four RrF1-subunits are expressed in the whole RrF1 molecule. Both antibodies agglutinated the R. rubrum chromatophores, suggesting that the beta-subunit is located on the external part of RrF1. 2. Both antibodies inhibited ATP synthesis and hydrolysis activities of R. rubrum chromatophores, as well as all the soluble ATPase reactions. Similar concentrations of each antibody were required for 50% inhibition of all these reactions, but anti-RrF1 was always somewhat more effective than anti-beta. These data indicate that the beta-subunit is involved in the catalytic site of the RrF1-enzyme. 3. The antibodies prepared against R. rubrum F1-ATPase and its beta-subunit could bind the soluble chloroplast F1-ATPase (CF1) and inhibited ATP-linked reactions carried out by chloroplasts and by soluble CF1. In these reactions, unlike in the R. rubrum ones, anti-beta was a more potent inhibitor than the anti-RrF1 antibody. The cross-reaction obtained between the antibodies raised against R. rubrum F1 and its beta-subunit and the chloroplast CF1 indicates the presence of similar antigenic determinants in the photosynthetic prokaryotic and eukaryotic F1-ATPases, which have been conserved during evolution.

Adenosine Triphosphatases↗

PubMed1996

Kinetic characterization of the hydrolytic activity of the H+-pyrophosphatase of Rhodospirillum rubrum in membrane-bound and isolated states.

Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1:1 and 1:2 . enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis.

Bacterial Proteins↗

PubMed1997

Sulfite stimulates the ATP hydrolysis activity of but not proton translocation by the ATP synthase of Rhodobacter capsulatus and interferes with its activation by delta muH+.

Sulfite stimulates the rate of ATP hydrolysis by the ATP synthase in chromatophores of Rhodobacter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in uncoupled chromatophores. The activation consists in an increase of about 12-15-fold of the Vmax for the ATP hydrolysis reaction, while the Km for MgATP is unaffected at 0.16+/-0.03 mM. The dependence of Vmax on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12 mM. Sulfite affects the ability of the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP-induced protonic flows demonstrate that at sulfite concentrations of greater than 10 mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by light or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentrations of at least 80 mM. Sulfite interferes with the mechanism of activation by delta muH+. Low concentrations of this anion (< or = 2 mM) prevent the activation by delta muH+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a delta muH+ across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite. These experimental results can be simulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K1S = 0.26 and K2S = 37 mM, and for phosphate K1P = 0.06 and K2P = 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is consistent with the molecular structures of the two ligands and of the enzyme.

Adenosine Triphosphate↗

PubMed1990

Isolation and characterization of a subunit form of the B875 light-harvesting complex from Rhodobacter capsulatus.

A structural subunit (called B816) has been isolated from the B875 light-harvesting complex of Rhodobacter capsulatus using a detergent-mediated dissociation of chromatophores. Rb. capsulatus MW442 (B800-850- B875+ car+) chromatophores were extracted with benzene and titrated with octyl glucoside (OG) to shift the near-infrared absorption maximum from 873 to 816 nm. Gel filtration chromatography was then used to separate B816 from reaction centers. B816 could be quantitatively shifted back to a B875-like form (lambda max = 875 nm) by decreasing the OG concentration. A similar B816 species could be isolated in low yield from wildtype (B800-850+ B875+ car+) cells but not from SB203E (B800-850- B875+ car-). In the latter case, the B816 subunit seemed too unstable to be isolated under equivalent conditions. The alpha:beta polypeptide ratio, the CD spectrum, and the ability to reversibly dissociate B816 to free bacteriochlorophyll and alpha- and beta-polypeptides were found to be similar to those of the B820 subunit of Rs. rubrum previously reported by our laboratory.

Bacterial Proteins↗