The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

Disruption of the Gene trx-m1 Impedes the Growth of Anabaena sp. PCC 7120 under Nitrogen Starvation.

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.

Development of heme protein based oxygen sensing indicators.

Oxygen is essential for aerobic life and is required for various oxygen-dependent biochemical reactions. In addition, oxygen plays important roles in multiple intracellular signaling pathways. Thus, to investigate oxygen homeostasis in living cells, we developed a genetically encoded oxygen sensor protein using the oxygen sensor domain of bacterial phosphodiesterase direct oxygen sensor protein (DosP), which was connected to yellow fluorescence protein (YFP) using an optimized antiparallel coiled-coil linker. The resulting ANA-Y (Anaerobic/aerobic sensing yellow fluorescence protein) was highly sensitive to oxygen and had a half saturation concentration of 18 µM. The ANA-Y reacts with dissolved oxygen within 10 s and the resulting increases in fluorescence are reversed with decreases in oxygen concentrations. This sensitivity of the ANA-Y enabled direct determinations of initial photosynthetic oxygen production by cyanobacteria. ANA-Y exhibits reversible fluorescence change of donor YFP following reversible absorbance change of acceptor DosH, and the operating mechanism of this ANA-Y could be used to develop various protein sensor probes for intracellular signaling molecules using natural sensor proteins.

Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO2 from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though Anabaena G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from Anabaena 7120 and its activator protein OpcA, we found that m-type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys393 and Cys399 of OpcA are responsible for the redox regulation property of this protein. Moreover, in vivo analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.

CyanoBase: a large-scale update on its 20th anniversary.

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN-BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e(-)) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase.

Rhodobacter sphaeroides mutants overexpressing chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways.

In previous studies we have demonstrated that chlorophyllide a oxidoreductases (CORs) from bacteriochlorophyll (BChl) a-producing Rhodobacter species and BChl b-producing Blastochloris viridis show distinct substrate recognition and different catalytic hydrogenation reactions, and that these two types of CORs therefore cause committed steps for BChls a and b biosynthesis. In this study, COR genes from B. viridis were incorporated and overexpressed in a series of Rhodobacter sphaeroides mutants. We found that the following two factors are essential in making R. sphaeroides produce BChl b: the loss of functions of both intrinsic COR and 8-vinyl reductase (BciA) in the host R. sphaeroides strain; and expression of the BchYZ catalytic components of COR from B. viridis, not the complete set of COR (BchXYZ), in the host strain. In addition, we incorporated bchYZ of B. viridis into the R. sphaeroides mutant lacking BchJ and BciA, resulting in the strain accumulating both BChl a and BChl b. This is the first example of an anoxygenic photosynthetic bacterium producing BChls a and b together. The results suggest that BchJ enhances activity of the intrinsic COR. The physiological significance of BchJ in pigment biosynthetic pathways will be discussed.

Involvement of thioredoxin on the scaffold activity of NifU in heterocyst cells of the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120.

The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA, 2001; 98: , 11224-11229)). Among these, the Fe-S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe-S cluster biogenesis.

Dark-operative protochlorophyllide oxidoreductase generates substrate radicals by an iron-sulphur cluster in bacteriochlorophyll biosynthesis.

Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms.

Nicotinamide is a specific inhibitor of dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, from Rhodobacter capsulatus.

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of two components, L-protein as a reductase component and NB-protein as a catalytic component. Elucidation of the crystal structures of NB-protein (Muraki et al., Nature 2010, 465: 110-114) has enabled us to study its reaction mechanism in combination with biochemical analysis. Here we demonstrate that nicotinamide (NA) inhibits DPOR activity by blocking the electron transfer from L-protein to NB-protein. A reaction scheme of DPOR, in which the binding of protochlorophyllide (Pchlide) to the NB-protein precedes the electron transfer from the L-protein, is proposed based on the NA effects.

An unexpectedly branched biosynthetic pathway for bacteriochlorophyll b capable of absorbing near-infrared light.

Chlorophyllous pigments are essential for photosynthesis. Bacteriochlorophyll (BChl) b has the characteristic C8-ethylidene group and therefore is the sole naturally occurring pigment having an absorption maximum at near-infrared light wavelength. Here we report that chlorophyllide a oxidoreductase (COR), a nitrogenase-like enzyme, showed distinct substrate recognition and catalytic reaction between BChl a- and b-producing proteobacteria. COR from BChl b-producing Blastochloris viridis synthesized the C8-ethylidene group from 8-vinyl-chlorophyllide a. In contrast, despite the highly conserved primary structures, COR from BChl a-producing Rhodobacter capsulatus catalyzes the C8-vinyl reduction as well as the previously known reaction of the C7 = C8 double bond reduction on 8-vinyl-chlorophyllide a. The present data indicate that the plasticity of the nitrogenase-like enzyme caused the branched pathways of BChls a and b biosynthesis, ultimately leading to ecologically different niches of BChl a- and b-based photosynthesis differentiated by more than 150 nm wavelength.

EPR study of 1Asp-3Cys ligated 4Fe-4S iron-sulfur cluster in NB-protein (BchN-BchB) of a dark-operative protochlorophyllide reductase complex.

Dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, contains two [4Fe-4S] clusters, one in the L-protein ((BchL)(2)) and the other in the NB-protein ((BchN-BchB)(2)). The reduced NB-cluster in the NB-protein, which is ligated by 1Asp/3Cys residues, showed a broad S=3/2 electron paramagnetic resonance signal that is rather rare in [4Fe-4S] clusters. A 4Cys-ligated NB-cluster in the mutated variant BchB-D36C protein, in which the Asp36 was replaced by a Cys, gave a rhombic normal S=1/2 signal and lost the catalytic activity. The results suggest that Asp36 contributes to the low redox potential necessary to reduce protochlorophyllide.

Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction.

Photosynthetic organisms require chlorophyll or bacteriochlorophyll for their light trapping and energy transduction activities. The biosynthetic pathways of chlorophyll and bacteriochlorophyll are similar in most of their early steps, except for the reduction of protochlorophyllide (Pchlide) to chlorophyllide. Whereas angiosperms make use of a light-dependent enzyme, cyanobacteria, algae, bryophytes, pteridophytes and gymnosperms contain an additional, light-independent enzyme dubbed dark-operative Pchlide oxidoreductase (DPOR). Anoxygenic photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides rely solely on DPOR. Recent atomic resolution of reductase and catalytic components of DPOR from R. sphaeroides and R. capsulatus, respectively, have revealed their similarity to nitrogenase components. In this review, we discuss the two fundamentally different mechanisms of Pchlide reduction in photosynthetic organisms.

X-ray crystal structure of the light-independent protochlorophyllide reductase.

Photosynthetic organisms adopt two different strategies for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide) to form chlorophyllide a, the direct precursor of chlorophyll a (refs 1-4). The first involves the activity of the light-dependent Pchlide oxidoreductase, and the second involves the light-independent (dark-operative) Pchlide oxidoreductase (DPOR). DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer), which are structurally related to nitrogenase Fe protein and MoFe protein, respectively. Here we report the crystal structure of the NB-protein of DPOR from Rhodobacter capsulatus at a resolution of 2.3A. As expected, the overall structure is similar to that of nitrogenase MoFe protein: each catalytic BchN-BchB unit contains one Pchlide and one iron-sulphur cluster (NB-cluster) coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific Pchlide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit. We propose a unique trans-specific reduction mechanism in which the distorted C17-propionate of Pchlide and an aspartate from BchB serve as proton donors for C18 and C17 of Pchlide, respectively. Intriguingly, the spatial arrangement of the NB-cluster and Pchlide is almost identical to that of the P-cluster and FeMo-cofactor in nitrogenase MoFe-protein, illustrating that a common architecture exists to reduce chemically stable multibonds of porphyrin and dinitrogen.

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis.

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto-plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast-chloroplast transition.

Functional expression of nitrogenase-like protochlorophyllide reductase from Rhodobacter capsulatus in Escherichia coli.

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction of protochlorophyllide in chlorophyll and bacteriochlorophyll biosynthesis. DPOR consists of two components, L-protein and NB-protein, which are structurally related to nitrogenase Fe-protein and MoFe-protein, respectively. Neither Fe-protein nor MoFe-protein is expressed as an active form in Escherichia coli due to the requirement of many Nif proteins for the assembly of the metallocenter and the maturation specific for diazotrophs. Here we report the functional expression of DPOR components from Rhodobacter capsulatus in Escherichia coli. Two overexpression plasmids for L-protein and NB-protein were constructed. L-protein and NB-protein purified from E. coli showed spectroscopic properties similar to those purified from R. capsulatus. L-protein and NB-protein activities were evaluated using a crude extract of E. coli overexpressing NB-protein and L-protein, respectively. Specific activities of the purified L-protein and NB-protein were 219+/-38 and 52.8+/-5.5 nmolChlorophyllide min(-1) mg(-1), respectively, which were even higher than those of L-protein and NB-protein purified from R. capsulatus. These E. coli strains provide a promising system for structural and kinetic analyses of the nitrogenase-like enzymes.

NB-protein (BchN-BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe-S clusters.

Dark-operative protochlorophyllide (Pchlide) oxidoreductase is a nitrogenase-like enzyme consisting of the two components, L-protein (BchL-dimer) and NB-protein (BchN-BchB-heterotetramer). Here, we show that NB-protein is the catalytic component with Fe-S clusters. NB-protein purified from Rhodobacter capsulatus bound Pchlide that was readily converted to chlorophyllide a upon the addition of L-protein and Mg-ATP. The activity of NB-protein was resistant to the exposure to air. A Pchlide-free form of NB-protein purified from a bchH-lacking mutant showed an absorption spectrum suggesting the presence of Fe-S centers. Together with the Fe and sulfide contents, these findings suggested that NB-protein carries two oxygen-tolerant [4Fe-4S] clusters.

Differential operation of dual protochlorophyllide reductases for chlorophyll biosynthesis in response to environmental oxygen levels in the cyanobacterium Leptolyngbya boryana.

Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO(2)/N(2)). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed.

Nitrogenase Fe protein-like Fe-S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide reductase (DPOR) in bacteriochlorophyll biosynthesis is a nitrogenase-like enzyme consisting of L-protein (BchL-dimer) as a reductase component and NB-protein (BchN-BchB-heterotetramer) as a catalytic component. Metallocenters of DPOR have not been identified. Here we report that L-protein has an oxygen-sensitive [4Fe-4S] cluster similar to nitrogenase Fe protein. Purified L-protein from Rhodobacter capsulatus showed absorption spectra and an electron paramagnetic resonance signal indicative of a [4Fe-4S] cluster. The activity quickly disappeared upon exposure to air with a half-life of 20s. These results suggest that the electron transfer mechanism is conserved in nitrogenase Fe protein and DPOR L-protein.

A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus.

In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.