Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans (original) (raw)
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Journal of Biological Chemistry, 2010
Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB) 2 , which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB) 2 (ChlL 2) 2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(,␥-imido)triphosphate, or MgADP in combination with AlF 4 ؊. Additionally, a mutant ChlL 2 protein, with a deleted Leu 153 in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL 2 , indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL 2 after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced.
Journal of Biological Chemistry, 2008
Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ring D by a protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL 2 and a heterotetrameric protein complex (ChlNB) 2. The ChlL 2 dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB) 2 complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB) 2. For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.
Reduction of Chemically Stable Multibonds: Nitrogenase-Like Biosynthesis of Tetrapyrroles
Advances in experimental medicine and biology, 2017
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...
Journal of Magnetic Resonance, Series B, 1996
Burkholderia cepacia AC1100 (formerly classified as a sulfur clusters, providing direct data about their type, oxidation state, and nearest surroundings (8, 9). Among the dis-Pseudomonas) degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide used in the Vietnam War for defolia-tinguishing characteristics of [2Fe-2S] clusters in the reduced state are the principal values of the g tensor. In all tion. A multiple component oxygenase is responsible for the conversion of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP) reported cases (10-14), the reduced Rieske clusters exhibit a characteristic g tensor (g 1 á 2.01-2.02, g 2 á 1.90-1.92, (1, 2). The genes encoding the oxygenase component have been cloned and sequenced (1). The oxygenase component g 3 á 1.76-1.8) with larger anisotropy and considerably lower g av á 1.91 than those of the plant-ferredoxin-type has a native molecular weight of 140,000, and it is composed of two 49 kDa a subunits and two 24 kDa b subunits. This cluster (g 1 á 2.04-2.05, g 2 á 1.96, g 3 á 1.87-1.88) or adrenal ferredoxin (g Å 2.025, g ⊥ Å 1.932) both with g av component is red and its spectrum in the visible region has maxima at 430 and 560 nm (shoulder), whereas upon reducá 1.96 (15, 16). Conclusive determination of the ligands in the Rieske-tion it has maxima at 420 (shoulder) and 530 nm. Each ab heterodimer contains three irons and two labile sulfides. type cluster was obtained by application of the solid-state, high-resolution EPR techniques of electron-nuclear double-These properties suggest the presence of a [2Fe-2S] cluster. At present two types of [2Fe-2S] clusters have been resonance (ENDOR) and electron-spin-echo envelope-modulation (ESEEM) spectroscopies. X-and Q-band ENDOR characterized. One is the widely distributed plant-ferredoxintype cluster where each iron atom is coordinated by the studies of the reduced Rieske-type cluster in phthalate dioxygenase from B. cepacia (17) and in ubiquinol-cytochrome sulfur atoms of two cysteine residues as shown in the available X-ray structures (3, 4). The other type of [2Fe-2S] c 2 oxidoreductase from Rhodobactor capsulatus (18) established that two histidine ligands are coordinated to one iron cluster with considerably higher redox potential was first described in the so-called Rieske protein isolated from the in place of two cysteines as in the plant ferredoxins and adrenodoxin. The ESEEM experiments performed on the ubiquinol-cytochrome c oxidoreductase of bovine heart mitochondria (5, 6). Similar proteins were found in other types reduced Rieske clusters in the cytochrome b 6 f complex of spinach chloroplast; in cytochrome bc 1 complexes of Rho-of membrane electron-transfer chains and aromatic dioxygenases (7). dospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria (19); and in Complex III of bovine Analysis of the deduced amino acid sequences of the a and b subunits in 2,4,5-T monooxygenase shows homology heart mitochondrial membranes (20) all show coordination with two histidine ligands. The reported values of hyperfine to the a and b subunits of other multicomponent aromatic dioxygenases. The a subunit has two conserved cysteine-constants of the two coordinated 14 N histidine nitrogens vary between 3.6 and 4.5 MHz and 4.6 and 5.5 MHz. In contrast, histidine pairs at the identical positions (Cys-X-His-17 amino acids-Cys-X-X-His) as in the other a subunits. This ENDOR and ESEEM investigations of plant-ferredoxin-type clusters show only interaction with nitrogens of the peptide motif is also repeated in Rieske iron-sulfur proteins. Electron paramagnetic resonance spectroscopy plays a backbone chain, with maximum hyperfine coupling Ç1 MHz (21-26). particularly important role in the characterization of iron-289
Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex
Proceedings of the National Academy of Sciences, 2013
Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a . The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride–stabilized (ADP·AlF 3 -stabilized) transition state complex between the DPOR components L 2 and (NB) 2 from the marine cyanobacterium Prochlorococcus marinus . Our analysis permits a thorough inves...
Journal of Biological Chemistry, 2006
In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A 696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A 696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A 696 formation and NADPH oxidation are identical, and we propose that formation of the A 696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the proS face of NADPH to the C-17 of Pchlide. The resulting A 696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction. The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR; EC 1.3.1.33) 2 catalyzes the reduction of the * This work was supported by the Joint Infrastructure Fund and the Biotechnology and Biological Sciences Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges.
Journal of Molecular Biology, 2010
Ferredoxin-NAD(P) + oxidoreductase (FNR) catalyzes the reduction of NAD(P) + to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to planttype FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structurefunction relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.
Biochemistry, 2004
The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP + cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP + from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.