Rhodobacter Capsulatus Mutants Research Articles

Two pathways to understanding electron transfer in reaction centers from photosynthetic bacteria: A comparison of Rhodobacter sphaeroides and Rhodobacter capsulatus mutants

The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P+HB− yields (up to ∼90%) than their R. capsulatus analogs (up to ∼60%), where HB is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near BB primarily increases the contribution of fast P* → P+BB− → P+HB− two-step ET, where BB is the “bridging” B-side bacteriochlorophyll. The second step (∼6–8 ps) is slower than the first (∼3–4 ps), unlike A-side two-step ET (P* → P+BA− → P+HA−) where the second step (∼1 ps) is faster than the first (∼3–4 ps) in the native RC. Substitutions near HB, at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20–50 ps) P* → P+HB− one-step superexchange ET. Both ET mechanisms are effective in directing electrons “the wrong way” to HB and both compete with internal conversion of P* to the ground state (∼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Photo-biological hydrogen production by a temperature-tolerant mutant of Rhodobacter capsulatus isolated by transposon mutagenesis

Screening of high temperature tolerant strains is important for photo-fermentative hydrogen production in natural conditions which exhibit wide temperature variations. Hence, a temperature-tolerant strain of Rhodobacter capsulatus was isolated by transposon mutagenesis. The mutant strain Rhodobacter capsulatus MX01 could convert cornstalk hydrolysate into hydrogen successfully, and exhibited better hydrogen production performance at higher culture temperature (33 °C and 37 °C) and light intensity (5000 lx and 7000 lx) than the wild type strain. At 33 °C and 5000 lx, the total hydrogen production yield and rate of MX01 from cornstalk hydrolysate were 3.64 ± 0.18 mol-H2/g-cornstalk and 40.07 ± 1.70 mmol-H2/(h·g-cornstalk), respectively. The energy conversion efficiency of cornstalk hydrolysate to hydrogen for the mutant strain MX01 was 10.6%. This higher temperature- and light intensity-tolerant mutant MX01 could carry out photo-fermentation at outdoor settings, which is important for eco-friendly, low-cost and energy-saving practical application of bio-hydrogen production.

Cu Transport by the Extended Family of CcoA-like Transporters (CalT) in Proteobacteria

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the “RfnT-like” CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb3-Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

High NH3[sbnd]N tolerance of a cheR2-deletion Rhodobacter capsulatus mutant for photo-fermentative hydrogen production using cornstalk

A mutant, JL1601, with a cheR2 gene deletion was constructed with Rhodobacter capsulatus JL1 as the wild type strain. The effects of pH, ammonia nitrogen concentration and light intensity on the photo-fermentative hydrogen production performance of JL1601 were studied. Suitable pH range for hydrogen production with the cheR2- mutant migrated from faintly acid to weak alkaline condition, and R.capsulatus JL1601 (cheR2-) showed higher NH3N tolerance. The mutant performed better H2 production performance than the wildtype under the same illumination condition. The maximum H2 yield and rate of R.capsulatus JL1601 (cheR2-) obtained to be 4406.7 ± 45.9 mL/L and 54.1 ± 1.6 mL/(L·h) with the mixture of acetate and butyrate as the carbon sources, which were increased by 36.3% and 12.3% in comparison to those of wildtype, respectively. Practical experiments using acid-enzymatic pretreated cornstalk as the carbon source indicated that, the maximum hydrogen yields by the mutant JL1601 (cheR2-) was up to 224.85 ± 5.18 mL-H2/g-cornstalk, which increased by 171.43% compared with that of wild type strain.

Primary processes in the bacterial reaction center probed by two-dimensional electronic spectroscopy

In the initial steps of photosynthesis, reaction centers convert solar energy to stable charge-separated states with near-unity quantum efficiency. The reaction center from purple bacteria remains an important model system for probing the structure-function relationship and understanding mechanisms of photosynthetic charge separation. Here we perform 2D electronic spectroscopy (2DES) on bacterial reaction centers (BRCs) from two mutants of the purple bacterium Rhodobacter capsulatus, spanning the Q y absorption bands of the BRC. We analyze the 2DES data using a multiexcitation global-fitting approach that employs a common set of basis spectra for all excitation frequencies, incorporating inputs from the linear absorption spectrum and the BRC structure. We extract the exciton energies, resolving the previously hidden upper exciton state of the special pair. We show that the time-dependent 2DES data are well-represented by a two-step sequential reaction scheme in which charge separation proceeds from the excited state of the special pair (P*) to P+HA- via the intermediate P+BA- When inhomogeneous broadening and Stark shifts of the B* band are taken into account we can adequately describe the 2DES data without the need to introduce a second charge-separation pathway originating from the excited state of the monomeric bacteriochlorophyll BA*.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus.

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA− mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb3-type cytochrome c oxidase (cbb3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb3-Cox production in the absence of DsbA. Indeed, addition of Cu2+ to the enriched medium suppresses, and conversely, omission of Cu2+ from the minimal medium induces, growth and cbb3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb3-Cox, especially in enriched medium where bioavailable Cu2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb3-Cox.

Enhanced biohydrogen production from cornstalk through a two-step fermentation: Dark fermentation and photofermentation

Summary The pretreated cornstalk (CS) was employed for biohydrogen production through combining dark-fermentative bacteria, cow dung, and photosynthetic bacteria, Rhodobacter capsulatus mutant in this work. In the first step, the cornstalk was pretreated with 0.75% NaOH, 12 IU/g-CS cellulase, and 2400 IU/g-CS hemicellulase under different hydrolysis time and temperature. The reducing sugar yields were very close under different conditions, and its maximum was 0.51 ± 0.01 g/g-CS at hydrolysis 108°C for 2.0 hours. However, the H2 yield of dark fermentation enhanced with the increasing hydrolysis time and temperature, and the maximum was 156.7 ± 8.6 mL/g-CS at hydrolysis 126°C for 2.0 hours. The biogas was only H2 and CO2, and no methane was found. In the second step, R. capsulatus ZYHAB3 with the mutation on both hvrA and hupAB genes was obtained from wild type (R. capsulatus SB1003) and the inactivation of both hvrA and hupAB genes remarkably enhanced nitrogenase activity. When the dark-fermentation effluent was employed as a substrate, the H2 yield and maximum H2 evolution rate of ZYHAB3 were 2827.5 ± 283.5 mL/L and 40.9 ± 2.3 mL/(Lh), which increased by 44.5 and 39.1% compared with those of wild type, respectively. The high H2 yield of 439.4 mL/g-CS was obtained from pretreated cornstalk through the 2-step process, and the chemical oxygen demand removal rate achieved 90.6%. The results suggest that combining cow dung and R. capsulatus mutant (hvrA− hupAB−) could be a promising way to produce H2 form agricultural wastes.

Comparative genomic analysis of two heat-resistant Rhodobacter capsulatus mutants with different hydrogen production levels reveals mutations related to hydrogen production

To overcome efficiency losses due to overheating in outdoor photobioreactors for microbial hydrogen production, heat-resistant microorganisms are desirable. In this study, the comparative whole genome sequencing analyses of two previously obtained Rhodobacter capsulatus heat-resistant mutants; A52 and B41, with modified hydrogen production capacities, have been performed to identify mutations related to hydrogen production. In comparison with the reference strain DSM1710, the genomes of the mutants A52 and B41 contained 2137 and 2253 mutations, respectively. In the mutant B41 genome, mutations were characterized within the nifD, nifJ, glnD, nifB1, ccpA, hupD, dmsA, and cbbR1 genes. On the other hand; in the A52 mutant, mutations were characterized in the nifB2, rnfF, nifJ, cbbO, anfH, amt, moeA, and hupD genes. The effects of nitrogen metabolism and redox-related mutations were tested by quantitative reverse transcription PCR. The most promising mutation was found on the glnD gene of B41 strain with higher hydrogen production capacity.

Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria

The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs “chlE.”

Improved photo – Hydrogen production by transposon mutant of Rhodobacter capsulatus with reduced pigment

The light shielding effect of photosynthetic bacteria in photo fermentation process, which is caused by high content of pigment, hinders their hydrogen production rates under intense light irradiation. In order to mitigate this effect and improve hydrogen production efficiency, it is necessary to screen mutants that hold less pigment content. In this study, a mini-Tn5 transposon encoded plasmid pRL27 was employed for transposon mutagenesis of Rhodobacter capsulatus SB1003. A mutant named MC1417 showed significant lower light absorbance from 330 nm to 900 nm compared to its parental strain by UV–visible spectra, and its bacteriochlorophyll a content was reduced by 38%. The results showed that its photo fermentative hydrogen production was improved by 50.5% on the basis of BChl a content using acetate and butyrate as carbon source under intense light irradiation, indicating that it is effective on improving hydrogen production by repressing the pigment biosynthesis. DNA sequencing and BLAST in NCBI Genebank showed that the mutation occurred within its pucDE gene.

Temperature resistant mutants of Rhodobacter capsulatus generated by a directed evolution approach and effects of temperature resistance on hydrogen production

Abstract Hydrogen (H2) is a promising alternative energy carrier which can be produced biologically. Rhodobacter capsulatus, a non-sulfur purple photosynthetic bacterium, can produce H2 under nitrogen-limited, photoheterotrophic conditions by using reduced carbon sources such as simple organic acids. Outdoor closed photobioreactors; used for biological H2 production are located under direct sunlight, as a result; bioreactors are exposed to temperature fluctuations during day time. In this study to overcome this problem, temperature-resistant mutants (up to 42 °C) of R. capsulatus were generated in this study by a directed evolution approach. Eleven mutant strains of R. capsulatus DSM 1710 were obtained by initial ethyl methane sulfonate (EMS) mutagenesis of the wild-type strain, followed by batch selection at gradually increasing temperatures up to 42 °C under respiratory conditions. The genetic stability of the mutants was tested and eight were genetically stable. Moreover, H2 production of mutant strains was analyzed; five mutants produced higher amounts of H2 when compared to the DSM 1710 wild-type strain and three mutants produced less H2 by volume. The highest H2- producing mutant (B41) produced 24% more H2 compared to wild type, and the mutant with lowest H2-production capacity (A52) generated 7% less H2 compared to the wild type. These results indicated that heat resistance of R. capsulatus can be improved by directed evolution, which is a useful tool to improve industrially important microbial properties. To understand molecular changes that confer high temperature-resistance and high hydrogen production capacity to these mutants, detailed transcriptomic and proteomic analyses would be necessary.

Biohydrogen production by Rhodobacter capsulatus Hup− mutant in pilot solar tubular photobioreactor

In this study, a pilot solar tubular photobioreactor was successfully implemented for fed batch operation in outdoor conditions for photofermentative hydrogen production with Rhodobacter capsulatus (Hup−) mutant. The bacteria had a rapid growth with a specific growth rate of 0.052 h−1 in the batch exponential phase and cell dry weight remained in the range of 1–1.5 g/L throughout the fed batch operation. The feeding strategy was to keep acetic acid concentration in the photobioreactor at the range of 20 mM by adjusting feed acetate concentration. The maximum molar productivity obtained was 0.40 mol H2/(m3 h) and the yield obtained was 0.35 mol H2 per mole of acetic acid fed. Evolved gas contained 95–99% hydrogen and the rest was carbon dioxide by volume.

Influence of the electric field on the electron transport in photosynthetic reaction centers

The effect of an electric field on the electron transfer in the bacterial reaction centers is investigated. The rate constants and quantum yields affected by the electric field for wild type (WT) and reaction center (RC) mutant of Rhodobacter capsulatus were computed. The dependence of the asymmetry of electron transfer in electric field on the temperature was evaluated. We found stable electron transfer for WT of the reaction center towards an electric field in comparison with the F(L121)D mutant of RC. We found quantum yields sensitive to the variation of the medium reorganization energy at low temperatures and strong electric fields. The quantum yields for unoriented RC samples were also calculated.

A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions.

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.

Trapping the P+BL− Initial Intermediate State of Charge Separation in Photosynthetic Reaction Centers from Rhodobacter capsulatus

The short-lived (<1 ps) initial intermediate state P(+)B(L)(-) in the photoinduced charge separation process of the bacterial photosynthetic reaction center has been trapped in two D(LL)-based Rhodobacter capsulatus mutants that have Tyr at position M208 and lack the bacteriopheophytin electron acceptor H(L). Transient state P(+)B(L)(-) is characterized by a 1017 nm bacteriochlorophyll anion absorption band and decays by charge recombination with a lifetime of several hundred picoseconds at 295 K. P(+)B(L)(-) is not observed in an otherwise identical mutant that has Phe at M208, which appears to make the state thermodynamically inaccessible from the excited primary electron donor P*.

Low-Temperature Studies of Electron Transfer to the M Side of YFH Reaction Centers from Rhodobacter capsulatus

There have been extensive experimental and theoretical studies of the temperature dependence of the rates of electron transfer between the cofactors associated primarily with the L polypeptide (or A branch) in the bacterial photosynthetic reaction center (RC). The focus of this paper is to gain further insight into the temperature dependence of rate of initial electron transfer to the parallel cofactor chain associated mainly with the M polypeptide (or B branch), which is inactive in the native RC. To this end, picosecond transient absorption measurements have been carried out on RCs of the YFH mutant of the photosynthetic bacterium Rhodobacter capsulatus at 77 K. In wild-type RCs, symmetry-related residues Phe M208 and Tyr L181 flank the primary electron donor (P) and are key to initial charge separation. In the YFH mutant these residues are swapped, i.e., are Tyr M208 and Phe L181. The third mutation in YFH changes Leu M212 to His and results in replacement of the L-side bacteriopheophytin (H(L)) with a bacteriochlorophyll denoted beta. Studies were carried out at 77 K for RCs in detergent-buffer/glycerol glasses utilizing either the detergent N-lauryl-N,N-dimethylamine N-oxide (LDAO) or Deriphat 160-C. In both media, excitation of P to its lowest singlet excited state (P*) elicits complex kinetic behavior that, in the simplest phenomenological description, involves two P* cofactor-protein populations: one that is capable of charge separation (active) and one that is not (inactive). The amplitudes of the components of the P* stimulated-emission kinetic profiles, and the amplitudes and time course of accompanying P-bleaching recovery, reveal that the two P* populations, active/inactive, are in 60/40 (LDAO) or 40/60 (Deriphat) proportion. In the nonphotoactive fraction, P* decays solely via return to the ground state with its inherent lifetime (i.e., lifetime in the absence of electron transfer) of 170 ps (LDAO) or 350 ps (Deriphat). In the photoactive fraction, P* has a lifetime of 4.5 ps (LDAO) or 13 ps (Deriphat) and decays by parallel electron transfer to H(M) (30%) and beta (70%) on the M and L branches, respectively. The rate constant for P* --> P(+)H(M)(-) electron transfer is (15 ps)(-1) (LDAO) or (43 ps)(-1) (Deriphat) at 77 K. These rate constants are about 2-fold greater than those determined at 295 K in the corresponding detergent/buffer solutions. These results combined with related findings from prior work show that the primary charge-separation events on both sides of the RC are basically activationless processes. In particular, in the functionally active P* population of YFH RCs, there is little or no apparent energy barrier (e.g., involving motions of the cofactors or protein or both) for electron transfer from P* to the either side of the RC. This conclusion holds irrespective of the precise description of the complex kinetic behavior that is observed. In addition to the observations on the temperature-dependent photochemistry, the 77 K transient absorption spectra in the near-infrared resolve a bacteriopheophytin anion band at 955 nm for wild-type RCs and, for the beta-containing mutants L(M212)H and YFH, a bacteriochlorophyll anion band at 1015 nm.

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the D(LL) mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the D(LL)-FY(L)F(M) mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845-3851). For D(LL), depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D(LL) RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D(LL)-FY(L)F(M) in Deriphat-glycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D(LL)-FY(L)F(M) RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of approximately 400 ps, similar to the deactivation of P* in the D(LL) mutant at 77 K. In the other 50% of D(LL)-FY(L)F(M) RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P+HM- in high yield (> or =80%). This result indicates that P* --> P(+)H(M)(-) is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* --> P(+)H(L)(-) in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* --> P(+)H(M)(-) is an activationless process.

Characterization of a Plant-like Protochlorophyllide a Divinyl Reductase in Green Sulfur Bacteria

The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur.

Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains

In Rhodobacter capsulatus excess reducing equivalents generated by organic acid oxidation is consumed to reduce protons into hydrogen by the activity of nitrogenase. Nitrogenase serves as a redox-balancing tool and is activated by the RegB/RegA global regulatory system during photosynthetic growth. The terminal cytochrome cbb 3 oxidase and the redox state of the cyclic photosynthetic electron transfer chain serve redox signaling to the RegB/RegA regulatory systems in Rhodobacter. In this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes or lacking the cyt cbb 3 oxidase or the quinol oxidase were compared with the wild type. The results indicated that hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3- to 4-fold, but the rate of hydrogen production increased significantly in a cbb 3 - mutant. Moreover, hydrogen production efficiency of various R. capsulatus strains further increased by inactivation of uptake hydrogenase genes.