Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Organization in photosynthetic membranes of purple bacteria in vivo: The role of carotenoids

Photosynthesis in purple bacteria is performed by pigment–protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC–LH1 pigment–protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC–LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions. However, mostly monomeric RC–LH1 complexes were isolated from strains expressing the native photoprotective red carotenoid spheroidenone, which is synthesized during phototrophic growth in the presence of oxygen. Despite this marked difference, linear dichroism (LD) and light-minus-dark LD spectra of oriented intact intracytoplasmic membranes indicated that RC–LH1 complexes are always assembled in ordered arrays, irrespective of variations in the relative amounts of isolated dimeric and monomeric RC–LH1 complexes. We propose that part of the photoprotective response to the presence of oxygen mediated by synthesis of spheroidenone may be a switch of the structure of the RC–LH1 complex from dimers to monomers, but that these monomers are still organized into the photosynthetic membrane in ordered arrays. When levels of the dimeric RC–LH1 complex were very high, and in the absence of LH2, LD and ?LD spectra from intact cells indicated an ordered arrangement of RC–LH1 complexes. Such a degree of ordering implies the presence of highly elongated, tubular membranes with dimensions requiring orientation along the length of the cell and in a proportion larger than previously observed.     

Replacement or exclusion of the B-branch bacteriopheophytin in the purple bacterial reaction centre: The HB cofactor is not required for assembly or core function of the Rhodobacter sphaeroides complex

All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the bacteriopheophytin (H_B) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the H_B bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the H_B position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the A-branch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an H_B bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the H_B cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (Q_B) site. The data are discussed with respect to possible functional roles of the H_B bacteriopheophytin, and we conclude that the reason(s) for conservation of a bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 < pH < 9.0, P⁺Q_B⁻ recombines with a pH independent average rate constant <k> more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which <k> increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH > 11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH > 9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q_AQ_B⁻ state is stabilized by about 40 meV at 6.5 < pH < 9.0, while it is destabilized at pH > 11. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and ³¹P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

Dimerisation of the Rhodobacter sphaeroides RC-LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide

In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC-LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC-LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC-LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC-LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC-LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC-LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH₂) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc₁ (cytbc₁) complexes that oxidise QH₂ to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc₁ complexes with gold nanobeads, each attached by a Histidine₁₀ (His₁₀)-tag to the C-terminus of cytc₁. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc₁ complexes occur as dimers in the membrane. The cytbc₁ complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc₁ complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc₁ whereas LH2 complexes became detached, consistent with clusters of cytbc₁ complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc₁–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc₁, ATP synthase, cytaa₃ and cytcbb₃ membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc₁ dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.     

Spectral dependence of energy transfer in wild-type peripheral light-harvesting complexes of photosynthetic bacteria

The precise position of the upper exciton component and relevant vibronic transitions of the B850 ring in peripheral light-harvesting complexes from purple photosynthetic bacteria are important values for determining the exciton bandwidth and electronic structure of the B850 ring. To determine the presence of these components in wild-type LH2 complexes the pump-probe femtosecond transient spectra obtained with excitation into the 730-840 nm spectral range are analyzed. We show that at excitation wavelengths less than 780 nm B850 absorption bands are present and that, in accordance with exciton theory, these bands peak further in the blue when the lowest optically allowed transition is more red-shifted.     

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm (Rps. sphaeroides) and 810–890 nm (Rps. viridis). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit

In this work, we report the unique case of bacteriochlorophyll (BChl) - protein covalent attachment in a photosynthetic membrane complex caused by a single mutation. The isoleucine L177 was substituted by histidine in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Pigment analysis revealed that one BChl molecule was missing in the acetone-methanol extract of the I(L177)H RCs. SDS-PAGE demonstrated that this BChl molecule could not be extracted with organic solvents apparently because of its stable covalent attachment to the mutant RC L-subunit. Our data indicate that the attached bacteriochlorophyll is one of the special pair BChls, P(A). The chemical nature of this covalent interaction remains to be identified.     

Analysis of absorption spectra of purple bacterial reaction centers in the near infrared region by higher order derivative spectroscopy

Reaction centers (RCs) of purple bacteria are uniquely suited objects to study the mechanisms of the photosynthetic conversion of light energy into chemical energy. A recently introduced method of higher order derivative spectroscopy [I.K. Mikhailyuk, H. Lokstein, A.P. Razjivin, A method of spectral subband decomposition by simultaneous fitting the initial spectrum and a set of its derivatives, J. Biochem. Biophys. Methods 63 (2005) 10-23] was used to analyze the NIR absorption spectra of RC preparations from Rhodobacter (R.) sphaeroides strain 2R and Blastochloris (B.) viridis strain KH, containing bacteriochlorophyll (BChl) a and b, respectively. Q(y) bands of individual RC porphyrin components (BChls and bacteriopheophytins, BPheo) were identified. The results indicate that the upper exciton level P(y+) of the photo-active BChl dimer in RCs of R. sphaeroides has an absorption maximum of 810nm. The blue shift of a complex integral band at approximately 800nm upon oxidation of the RC is caused primarily by bleaching of P(y+), rather than by an electrochromic shift of the absorption band(s) of the monomeric BChls. Likewise, the disappearance of a band peaking at 842nm upon oxidation of RCs from B. viridis indicates that this band has to be assigned to P(y+). A blue shift of an absorption band at approximately 830nm upon oxidation of RCs of B. viridis is also essentially caused by the disappearance of P(y+), rather than by an electrochromic shift of the absorption bands of monomeric BChls. Absorption maxima of the monomeric BChls, B(B) and B(A) are at 802 and 797nm, respectively, in RCs of R. sphaeroides at room temperature. BPheo co-factors H(B) and H(A) peak at 748 and 758nm, respectively, at room temperature. For B. viridis RCs the spectral positions of H(B) and H(A) were found to be 796 and 816nm, respectively, at room temperature.     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q_A⁻Q_B→Q_AQ_B⁻ (k_AB⁽¹⁾) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k_AB⁽¹⁾, attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q_B [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm⁻¹, characteristic of Q_A reduction. The time evolution of the spectra shows reoxidation of Q_A⁻ and concomitant reduction of Q_B with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q_B⁻ occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm⁻¹ [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q_B in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q_A⁻ to Q_B and the identification of Glu-L212 as the main proton acceptor in the state Q_AQ_B⁻.     

Defective assembly of a hybrid vacuolar H-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus

Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2′-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E. coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. It appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus.