Dark-operative Protochlorophyllide Oxidoreductase Research Articles

Alternative Electron Donors for the Nitrogenase‐like Dark‐Operative Protochlorophyllide Oxidoreductase (DPOR)

AbstractSharing structural and mechanistic properties with the dinitrogen‐fixing enzyme nitrogenase, dark‐operative protochlorophyllide oxidoreductase (DPOR) participates in the biosynthesis of bacteriochlorophyll, subsequently enabling photosynthesis by photosynthetic bacteria. In contrast to the light‐dependent POR, electron transfer in DPOR is coupled to the hydrolysis of adenosine triphosphate for the two‐electron stereoselective reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a coupling also employed by nitrogenases for the multielectron reduction of dinitrogen to ammonia. The importance of adenosine triphosphate hydrolysis is not fully understood for these enzymes, and electrochemistry is a valuable technique to regulate and/or monitor electron delivery to these enzymes. Here, we report three alternative electron donors that support protochlorophyllide reduction in the DPOR system, which could also enable electroenzymatic studies of DPOR. We demonstrate the turnover of DPOR within a spectroelectrochemical configuration, permitting the formation of Chlide to be followed and coupled to electron utilization by DPOR.

Single-molecule studies of asymmetric substrate binding and electron transfer in the nitrogenase-like DPOR complex

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an A2B2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear.

The roles of a light-dependent protochlorophyllide oxidoreductase (LPOR), and ATP-dependent dark operative protochlorophyllide oxidoreductase (DPOR) in chlorophyll biosynthesis

Chlorophyll is a green photosynthetic pigment, and photosynthesis drives the global carbon cycle. The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in the penultimate stage of biosynthesis of chlorophyll (Chl) is catalyzed by light-independent protochlorophyllide reducatse (DPOR), and the light-dependent protochlorophyllide oxidoreductase (LPOR). The search was done to all manuscript sections according to terms chlorophyll, a light-dependent protochlorophyllide oxidoreductase, ATP-dependent dark operative protochlorophyllide oxidoreductase, chlorophyll, photosynthesis and chlorophyllide. Within the framework of photosynthesis and chlorophyll, this review article was aimed to provide an overview of the functional studies in chlorophyll biosynthesis, protein crystal structure, disclosure of action mechanisms, and possible future available direction of LPOR and DPOR in the biosynthesis of chlorophyll.

The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]–containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen–deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Methods for Heterologous Overproduction of Fe-S Proteins.

Proteins carrying iron-sulfur ([Fe-S]) clusters are critical to the basic metabolism of all organisms. Structural and biochemical investigations of many such [Fe-S] cluster proteins depend on recombinant overproduction using heterologous bacterial hosts such as Escherichia coli . Here, we describe a detailed procedure for the overproduction and purification of two oxygen-sensitive component proteins of the dark-operative protochlorophyllide oxidoreductase (DPOR) complex. The method relies on an engineered Escherichia coli cell line carrying a correction in its genome to restore the loss of a key [Fe-S] cluster biogenesis pathway. The method can also be potentially adapted for the overproduction of other Fe-S proteins.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

Substrate recognition induces sequential electron transfer across subunits in the nitrogenase-like DPOR complex

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α2β2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

Dark chlorophyll synthesis may provide a potential for shade tolerance as shown by a comparative study with seedlings of European larch (Larix decidua) and Norway spruce (Picea abies)

The ability to green in the dark represents a trait providing shade tolerance in seedlings of Norway spruce. In contrast to angiosperm plants, gymnosperms possess two different enzymes for the reduction of protochlorophyllide to chlorophyllide: dark-operative, light-independent protochlorophyllide oxidoreductase (DPOR), consisting of three subunits: ChlL, ChlN and ChlB, and light-dependent protochlorophyllide oxidoreductase (LPOR). European larch seedlings (Larix decidua Mill.), in contrast to Norway spruce (Picea abies Karst.), accumulate only very low amounts of chlorophylls in the dark due to an inactive DPOR enzyme. In this study, we used these two species to investigate the advantage of the co-existence of two protochlorophyllide oxidoreductases on chlorophyll synthesis under different light conditions. We found that under deep shade conditions, the larch seedlings are partially etiolated with a low quantum yield of photosystem II photochemistry caused by inefficient LPOR function under that subliminal irradiance concomitant with an inactive DPOR enzyme. In contrast, spruce accumulated a significant amount of chlorophylls under deep shade conditions due to the co-existence of active DPOR and LPOR enzymes. Moreover, although PSII developed in the dark had an inactive oxygen-evolving complex, even very low irradiance is sufficient for photoactivation of PSII, as proved by the high quantum yield of photosystem II (Fv/Fm) and the disappearance of the K-step in chlorophyll a fluorescence induction under deep shade conditions in spruce. We did not find any advantage of having DPOR enzyme under high light conditions, which is consistent with the decreasing abundance of DPOR subunits with increasing light intensities. Thus, the presence of an active DPOR enzyme may represent a molecular basis for shade tolerance in coniferous seedlings.

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Erratum to: Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7 °C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7°C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

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.

Reduction of Chemically Stable Multibonds: Nitrogenase-Like Biosynthesis of Tetrapyrroles.

The sophisticated biochemistry of nitrogenase plays a fundamental role for the biosynthesis of tetrapyrrole molecules, acting as key components of photosynthesis and methanogenesis. Three nitrogenase-like metalloenzymes have been characterized to date. Synthesis of chlorophylls and bacteriochlorophylls involves the reduction of the C17-C18 double bond of the conjugated ring system of protochlorophyllide which is catalyzed by the multi-subunit enzyme dark operative protochlorophyllide oxidoreductase (DPOR). Subsequently, biosynthesis of all bacteriochlorophylls requires the reduction of the C7-C8 double bond by a second nitrogenase-like enzyme termed chlorophyllide oxidoreductase (COR). Mechanistically, DPOR and COR make use of a reductase component which links ATP hydrolysis to conformational changes. This dynamic switch protein is triggering the transient association between the reductase and the core catalytic protein complex, thereby facilitating the transduction of electrons via two [4Fe4S] clusters. X-ray crystallographic structural investigations in combination with biochemical experiments revealed the molecular basis of the underlying energy transduction mechanism. The unique nickel-containing tetrapyrrole cofactor F430 is located in the active site of methyl-coenzyme M reductase, which is catalyzing the final step of methane formation in methanogenic archaea. The nitrogenase-like protein NflH/NflD has been proposed to catalyze one or more ring reduction steps during the biosynthesis of F430. The present working hypothesis mirrors a DPOR and COR related enzyme mechanism of NflH/NflD. Furthermore, nfl-encoded proteins were suggested as "simplified" ancestors lying basal in the phylogenetic tree between nitrogenase and DPOR/COR.

Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD(+) complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD(+) as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.

With or without light: comparing the reaction mechanism of dark-operative protochlorophyllide oxidoreductase with the energetic requirements of the light-dependent protochlorophyllide oxidoreductase.

The addition of two electrons and two protons to the C17=C18 bond in protochlorophyllide is catalyzed by a light-dependent enzyme relying on NADPH as electron donor, and by a light-independent enzyme bearing a (Cys)3Asp-ligated [4Fe–4S] cluster which is reduced by cytoplasmic electron donors in an ATP-dependent manner and then functions as electron donor to protochlorophyllide. The precise sequence of events occurring at the C17=C18 bond has not, however, been determined experimentally in the dark-operating enzyme. In this paper, we present the computational investigation of the reaction mechanism of this enzyme at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level of theory. The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe–4S], yielding a negatively-charged intermediate. Depending on the rate of Fe–S cluster re-reduction, the reaction either proceeds through double protonation of the single-electron-reduced substrate, or by alternating proton/electron transfer. The computed reaction barriers suggest that Fe–S cluster re-reduction should be the rate-limiting stage of the process. Poisson–Boltzmann computations on the full enzyme–substrate complex, followed by Monte Carlo simulations of redox and protonation titrations revealed a hitherto unsuspected pH-dependence of the reaction potential of the Fe–S cluster. Furthermore, the computed distributions of protonation states of the His, Asp and Glu residues were used in conjuntion with single-point ONIOM computations to obtain, for the first time, the influence of all protonation states of an enzyme on the reaction it catalyzes. Despite exaggerating the ease of reduction of the substrate, these computations confirmed the broad features of the reaction mechanism obtained with the medium-sized models, and afforded valuable insights on the influence of the titratable amino acids on each reaction step. Additional comparisons of the energetic features of the reaction intermediates with those of common biochemical redox intermediates suggest a surprisingly simple explanation for the mechanistic differences between the dark-catalyzed and light-dependent enzyme reaction mechanisms.

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.

Reconstitution of a sequential reaction of two nitrogenase-like enzymes in the bacteriochlorophyll biosynthetic pathway of Rhodobacter capsulatus

The parental structure of bacteriochlorophyll a, bacteriochlorin, is formed by a sequential operation of two nitrogenase-like enzymes, dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide a oxidoreductase (COR). Both DPOR and COR consist of two components, Fe protein and MoFe protein cognates. Here we determined kinetic parameters of COR and established the reconstitution system for the formation of bacteriochlorin (3-vinyl bacteriochlorophyllide a) from porphyrin (protochlorophyllide) with purified components of DPOR and COR from Rhodobacter capsulatus. This reconstitution system confirmed the recent finding that COR catalyzes 8-vinyl reduction of 8-vinyl chlorophyllide a in addition to the known activity of C7C8 double bond reduction, and provides a promising model to investigate how two nitrogenase-like enzymes are coordinated in bacteriochlorophyll biosynthesis.

3:1] Site-Differentiated [4Fe–4S] Clusters Having One Carboxylate and Three Thiolates

[4Fe-4S] clusters modeled after those in organisms having three cysteine thiolates and one carboxylate were synthesized by using the tridentate thiolato chelate. X-ray structural analysis reveals that the carboxylates coordinate to the unique irons in an η(1) manner rather than η(2). Redox potentials show a positive shift from that of the cluster having ethanethiolate and the tridentate thiolato chelate. These properties conform to the arrangement of the [4Fe-4S] clusters in the electron transfer systems included in Rc dark operative protochlorophyllide oxidoreductase (DPOR) and formaldehyde oxidoreductase (FOR) with Pf ferredoxin.

Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes

High-quality NMR structures of the C-terminal domain comprising residues 484-537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9-61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/β fold comprised of three α-helices and a small β-sheet packed against second α-helix. These two proteins share 29% sequence similarity and their structures are globally quite similar. The structures of BchB(484-537) and Asr4154(9-61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484-537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.

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.