Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

DAC Is Involved in the Accumulation of the Cytochrome b6/f Complex in Arabidopsis

The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b₆/f complex, and provide evidence suggesting that the efficiency of cytochrome b₆/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b₆/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b₆/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b₆ protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b₆/f complex, possibly through interaction with the PetD protein.The cytochrome b₆/f (Cyt b6/f) complex is a multisubunit complex that resides in the thylakoid membrane and functions in linear and cyclic electron transport. In the linear process, the complex receives electrons from PSII and transfers them to PSI, a process that is accompanied by the generation of a proton gradient, which is essential for ATP synthesis (Mitchell, 1961; Saraste, 1999). The native form of this complex is present as a dimer with a mass of 310 kD that can be converted into a 140-kD monomer with increasing detergent concentrations (Huang et al., 1994; Breyton et al., 1997; Mosser et al., 1997; Baniulis et al., 2009). In higher plants, the Cyt b6/f monomer contains at least eight subunits: Cyt f, Cyt b₆, PetC, PetD, PetM, PetL, PetG, and PetN (Wollman, 2004). PetC and PetM are encoded by nuclear genes, whereas the others are encoded by plastid genes. It has been shown that PetG and PetN are necessary for complex stability in tobacco (Nicotiana tabacum; Schwenkert et al., 2007). By contrast, PetL is not required for the accumulation of other subunits of the Cyt b6/f complex, even though it is involved in the stability and formation of the functional dimer (Bendall et al., 1986; Schwenkert et al., 2007). Inactivation of PetC in Arabidopsis (Arabidopsis thaliana) resulted in significantly reduced amounts of Cyt b6/f subunits and completely blocked linear electron transport, indicating that PetC participates in the formation of the functionally assembled Cyt b6/f complex (Maiwald et al., 2003). In Synechocystis sp. PCC 6803, the PetM subunit has no essential role in Cyt b6/f complex electron transfer or accumulation; however, the absence of this subunit apparently affects the levels of other protein complexes involved in energy transduction (Schneider et al., 2001). In addition to the other proteins, FNR was identified as a subunit of the Cyt b6/f complex isolated from spinach (Spinacia oleracea) thylakoid membranes (Zhang et al., 2001).Previous research has revealed how the Cyt b6/f complex assembles into a functional dimer (Bendall et al., 1986; Lemaire et al., 1986; Kuras and Wollman, 1994). In the Cyt b6/f complex, Cyt b₆ and PetD form a mildly protease-resistant subcomplex that serves as a template for the assembly of Cyt f and PetG, producing a protease-resistant cytochrome moiety (Wollman, 2004). The PetC and PetL proteins then participate in the assembly of the functional dimer (Schwenkert et al., 2007). PetD becomes more unstable in the absence of Cyt b₆, and the synthesis of Cyt f is greatly reduced when either Cyt b₆ or PetD is inactivated, indicating that both Cyt b₆ and PetD are prerequisite for the synthesis of Cyt f (Kuras and Wollman, 1994). The reduced synthesis of Cyt f can be explained by the so-called CES (for controlled by epistasy of synthesis) mechanism. It is suggested that, in this mechanism, the synthesis rate of some chloroplast-encoded subunits of photosynthetic protein complexes is regulated by the availability of their assembly partners from the same complexes (Choquet et al., 2001). The mechanism of CES for Cyt f has been studied in detail in Chlamydomonas reinhardtii (Choquet et al., 1998; Choquet and Vallon, 2000). In it, the unassembled Cyt f inhibits its own translation through a negative feedback mechanism, and MCA1 and TCA1 have been demonstrated to be involved in the regulation of Cyt f synthesis (Boulouis et al., 2011).Many studies have focused on understanding the conversion of apocytochrome to holocytochrome via the covalent binding of heme in Cyt f and Cyt b₆ during the assembly of Cyt b6/f through the CCS and CCB pathways (Nakamoto et al., 2000; Wollman, 2004; de Vitry, 2011). The CCS pathway was originally discovered in the green alga C. reinhardtii through genetic studies of ccs mutants (for cytochrome c synthesis) that display a specific defect in membrane-bound Cyt f and soluble Cyt c₆, two thylakoid lumen-resident c-type cytochromes functioning in photosynthesis (Xie and Merchant, 1998). In the CCS pathway, six loci that include plastid ccsA and nuclear CCS1 to CCS5 have been found in C. reinhardtii (Xie and Merchant, 1998). In these mutants, the apocytochrome is normally synthesized, targeted, and processed, but heme attachment is perturbed. The CCB pathway is involved in the covalent attachment of heme c(i) to Cyt b₆ on the stromal side of the thylakoid membranes (Kuras et al., 2007). The ccb mutants show defects in the accumulation of subunits of the Cyt b6/f complex and covalent binding of heme to Cyt b₆ (Lyska et al., 2007; Lezhneva et al., 2008). However, heme binding is not a prerequisite for the assembly of Cyt b₆ into the Cyt b6/f complex, although the fully formed Cyt b6/f showed an increased sensitivity to protease (Saint-Marcoux et al., 2009).The assembly of the Cyt b6/f complex is a multistep process, and current studies have shown that the covalent binding of heme to Cyt f and Cyt b₆ is highly regulated. Thus, it is reasonable to speculate that, similar to the other photosynthetic protein complexes (Mulo et al., 2008; Nixon et al., 2010; Rochaix, 2011), the assembly of the Cyt b6/f complex is also assisted by many nucleus-encoded factors. In this study, we characterized an Arabidopsis protein, DAC (for defective accumulation of Cyt b6/f complex), that seems to be involved in the assembly of the Cyt b6/f complex. In addition, we provide evidence that DAC interacts directly with PetD before it assembles within the Cyt b6/f complex.     

Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.     

The cytochrome bc1 complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc1.

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.     

Cryo-EM structure of a monomeric RC-LH1-PufX supercomplex with high-carotenoid content from Rhodobacter capsulatus

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Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b₆/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b₆, and Rieske Fe-S center protein in cytochrome b₆/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b₆/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b₆, and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.     

An optimized g-tensor for Rhodobacter capsulatus cytochrome c2 in solution: a structural comparison of the reduced and oxidized states.

The optimized g-tensor parameters for the oxidized form of Rhodobacter capsulatus cytochrome c2 in solution were obtained using a set (50) of backbone amide protons. Dipolar shifts for more than 500 individual protons of R. capsulatus cytochrome c2 have been calculated by using the optimized g-tensor and the X-ray crystallographic coordinates of the reduced form of R. capsulatus cytochrome c2. The calculated results for dipolar shifts are compared with the observed paramagnetic shifts. The calculated and the observed data are in good agreement throughout the entire protein, but there are significant differences between calculated and experimental results localized to the regions in the immediate vicinity of the heme ligand and the region of the front crevice of the protein (residues 44-50, 53-57, and 61-68). The results not only indicate that the overall solution structures are very similar in both the reduced and oxidized states, but that these structures in solution are similar to the crystal structure. However, there are small structural changes near the heme and the rearrangement of certain residues that result in changes in their hydrogen bonding concomitant with the change in the oxidation states; this was also evident in the data for the NH exchange rate measurements for R. capsulatus cytochrome c2.     

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

The Isolation of a Functional Cytochrome b 6 f Complex: from Lucky Encounter to Rewarding Experiences

The recognition that, in photosynthesis, the plastoquinol oxidizing cytochrome b (6f ) complex resembles the ubiquinol oxidizing cytochrome bc1 complex in respiration is one of the examples of exciting universalization in biological research. A peripheral observation towards the end of 1979 initiated an intensive investigation, which is still ongoing today: next to the ATP synthase the cytochrome b (6f ) complex could be selectively solubilized from the chloroplast membrane by the combined action of octyl glucoside and cholate. It was mere luck that the isolate was substantially active as an electrogenic, proton translocating plastoquinol-plastocyanin oxidoreductase, and that it also catalyzed oxidant-induced reduction of cytochrome b (6), a signature of the Q-cycle mechanism. The basic findings during the first characterization of the complex are summarized, and the excitement among the collaborating groups is remembered. More recent developments, including the impact of gene technology and the elucidation by the crystal structure, are additionally traced here.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide

Cytochrome bc₁ is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Q_o site in cytochrome b with cytochrome c₁. The Q_o site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Q_o site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Q_o site. This renders cytochrome bc₁ non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Q_o site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc₁ to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which “semireverse” electron transfer back to the Q_o site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: II. KINETICS AND MECHANISM OF BINDING*

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c₁ of detergent-solubilized bc₁ complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c₁-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mm Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mm, the rate leveled off, indicating a rate-limiting conformational step with lifetime ∼1 s; and (iii) at Im concentrations above 100 mm, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.     

HCF164 Encodes a Thioredoxin-Like Protein Involved in the Biogenesis of the Cytochrome b6f Complex in Arabidopsis

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.     

Streptococcus thermophilus Is Able To Produce a β-Galactosidase Active during Its Transit in the Digestive Tract of Germ-Free Mice

This work presents data on the application of a bacterial luciferase used to monitor gene expression of Streptococcus thermophilus in the digestive tract. The main result is that the bacterium was able to produce an active β-galactosidase in the digestive tract, although it did not multiply during its transit. This production was enhanced when lactose (the inducer) was added to the diet.     

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

Effect of Mutations of Arginine 94 on Proton Pumping,Electron Transfer,and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc₁ complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc₁ complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O₂^˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc₁ complex in R. sphaeroides. This also suggests that the transport of H⁺, O₂, and O₂^˙̄ in the bc₁ complex may occur by the same pathway.     

The PuhB protein of Rhodobacter capsulatus functions in photosynthetic reaction center assembly with a secondary effect on light-harvesting complex 1

The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.     

Rhizobium etli USDA9032 Engineered To Produce a Phenazine Antibiotic Inhibits the Growth of Fungal Pathogens but Is Impaired in Symbiotic Performance

Phenazine production was engineered in Rhizobium etli USDA9032 by the introduction of the phz locus of Pseudomonas chlororaphis O6. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish Fix⁻ nodules.     

Reduction of Hg2+ with Reduced Mammalian Cytochrome c by Cytochrome c Oxidase Purified from a Mercury-Resistant Acidithiobacillus ferrooxidans Strain,MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 μM and 5 μM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 μM, and 70% by 10 μM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.