RESUMEN
Reduction of the 17,18-double bond in the D-ring during chlorophyll biosynthesis is catalyzed by the rare, naturally occurring photoenzyme protochlorophyllide oxidoreductase (POR). A conserved tyrosine residue has been suggested to donate a proton to C18 of the substrate in the past decades. Taylor and colleagues scrutinized the model with a powerful tool that utilized a modified genetic code to introduce fluorinated tyrosine analogues into POR. The presented results show that the suggested catalytically critical tyrosine is unlikely to participate in the reaction chemistry but is required for substrate binding, and instead, a cysteine residue preceding the lid helix is proposed to have the role of proton donor.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Protoclorofilida , Halogenación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/química , Protones , Clorofila/biosíntesis , Clorofila/metabolismoRESUMEN
The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.
Asunto(s)
Cianobacterias , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Protoclorofilida/química , Luz , Protones , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , FotoquímicaRESUMEN
Protochlorophyllide(PChlide)-a and its 8-vinylated analog, divinyl(DV)-PChlide-a, are common and essential intermediates in the biosynthesis of all naturally occurring chlorophyll (Chl) pigments. These porphyrinoid-type pigments have a single optically active (asymmetric) carbon atom at the 132-position, so their stereoisomers are (132R)- and (132S)-enantiomers. The former and latter are called (DV-)PChlide-a and (DV-)PChlide-a', respectively. In this study, chiral-phase HPLC separation of enantiomeric (DV-)PChlides-a/a' was demonstrated. The (132R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (132S)-enantiomeric PChlide-a' under the present HPLC conditions. On the other hand, the elution order of (132R)-DV-PChlide-a and (132S)-DV-PChlide-a' was reverse to that of PChlides-a/a'. After the separation of each enantiomer by the chiral-phase HPLC, the stereoisomeric configuration at the 132-position was characterized by means of circular dichroism spectroscopy. The present chiral-phase HPLC method enables us to evaluate optical purities of (DV-)PChlide-a species. For example, PChlide-a and/or DV-PChlide-a extracted from the spent medium and harvested cells of cultured purple photosynthetic bacterial mutants, the former of which has been often used as the source of (DV-)PChlide-a substrates for enzymatic reactions, were revealed to be mostly racemized, giving enantiomeric mixtures of (DV-)PChlides-a/a'.
Asunto(s)
Clorofila , Protoclorofilida , Protoclorofilida/química , Estereoisomerismo , Cromatografía Líquida de Alta Presión , Clorofila/químicaRESUMEN
NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. It is one of few known photoenzymes, which catalyzes the light-activated trans-reduction of the C17-C18 double bond of Pchlide's porphyrin ring. Due to the light requirement, dark-grown angiosperms cannot synthesize chlorophyll. No crystal structure of POR is available, so to improve understanding of the protein's three-dimensional structure, its dimerization, and binding of ligands (both the cofactor NADPH and substrate Pchlide), we computationally investigated the sequence and structural relationships among homologous proteins identified through database searches. The results indicate that α4 and α7 helices of monomers form the interface of POR dimers. On the basis of conserved residues, we predicted 11 functionally important amino acids that play important roles in POR binding to NADPH. Structural comparison of available crystal structures revealed that they participate in formation of binding pockets that accommodate the Pchlide ligand, and that five atoms of the closed tetrapyrrole are involved in non-bonding interactions. However, we detected no clear pattern in the physico-chemical characteristics of the amino acids they interact with. Thus, we hypothesize that interactions of these atoms in the Pchlide porphyrin ring are important to hold the ligand within the POR binding site. Analysis of Pchlide binding in POR by molecular docking and PELE simulations revealed that the orientation of the nicotinamide group is important for Pchlide binding. These findings highlight the complexity of interactions of porphyrin-containing ligands with proteins, and we suggest that fit-inducing processes play important roles in POR-Pchlide interactions.
Asunto(s)
NADP/metabolismo , Pisum sativum/metabolismo , Proteínas de Plantas , Protoclorofilida , Sitios de Unión , Dimerización , Ligandos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Protoclorofilida/química , Protoclorofilida/metabolismoRESUMEN
Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.
Asunto(s)
Clorofila/química , Clorofilidas/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Fotosíntesis/genética , Protoclorofilida/química , Secuencia de Aminoácidos , Dominio Catalítico , Clorofila/biosíntesis , Clorofilidas/biosíntesis , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , NADP/química , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Plantas/enzimología , Plantas/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Protoclorofilida/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermosynechococcus/enzimología , Thermosynechococcus/genéticaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Hierro-Azufre/metabolismo , Adenosina Trifosfato/química , Catálisis , Proteínas Hierro-Azufre/química , Espectrometría de Masas , Nitrogenasa/química , Nitrogenasa/metabolismo , Fotosíntesis , Protoclorofilida/química , Protoclorofilida/metabolismo , Especificidad por SustratoRESUMEN
The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structure-function relationships of the light-driven Pchlide reduction.
Asunto(s)
Cianobacterias/enzimología , Luz , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/metabolismo , Synechocystis/enzimología , Catálisis , Clorofila/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , NADP/química , Conformación Proteica , Protoclorofilida/química , Protones , ThermosynechococcusRESUMEN
Anthocyanins (Ants) are water-soluble secondary metabolites that are responsible for red colour of plant leaves. To determine photosynthetic pigments, 80% acetone was used to extract Ants from Ant-containing leaves of test plants. However, using the 80% acetone extraction method can lead to interference between chlorophylls (Chls) and Ants. Porphyrins, such as protoporphyrin IX (PPIX), Mg-protoporphyrin IX (MgPP), and protochlorophyllide (Pchlide), are Chl biosynthetic intermediates and demonstrate photospectrometric characteristics similar to those of Chl. Although the ether/water extraction method was able to remove Ants interference when detecting porphyrins, the porphyrins extraction efficiency was lower than that of the 80% acetone extraction method. Low Ants levels interfered with individual porphyrin ratios, and the extent of the effect was correlated with Ants concentrations. We developed the three equations could eliminate interference by Ants when determining the porphyrin molecular percentage (%) and were comprehensively applied to all tested species of Ants-containing leaves.
Asunto(s)
Antocianinas/metabolismo , Hojas de la Planta/química , Porfirinas/metabolismo , Antocianinas/química , Clorofila/biosíntesis , Color , Ipomoea batatas/química , Ipomoea batatas/metabolismo , Hojas de la Planta/metabolismo , Porfirinas/química , Protoclorofilida/química , Protoclorofilida/metabolismo , Protoporfirinas/química , Protoporfirinas/metabolismoRESUMEN
Hydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light-dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C17 -C18 double bond of protochlorophyllide. Time-resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton-coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C17 or C18 , but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.
Asunto(s)
Luz , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , NADP/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Protoclorofilida/química , Protoclorofilida/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Cloroplastos/enzimología , Cianobacterias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Pinus/enzimología , Proteínas de Plantas/química , Protoclorofilida/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cloroplastos/genética , Clonación Molecular , Cianobacterias/genética , Oscuridad , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fotosíntesis/genética , Pinus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Protoclorofilida/metabolismo , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
The chlorophyll precursor protochlorophyllide (Pchlide), which is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase, has unique excited-state properties that facilitate photocatalysis. Previous time-resolved spectroscopy measurements have implied that a long-lived triplet state is formed during the excited-state relaxation of Pchlide, although direct evidence of its existence is still lacking. Here we use time-resolved electron paramagnetic resonance (EPR) in combination with time-resolved absorption measurements at a range of temperatures (10-290 K), solvents, and oxygen concentrations to provide a detailed characterization of the triplet state of Pchlide. The triplet decays in a biphasic, oxygen-dependent manner, while the first reported EPR signature of a Pchlide triplet displays both emissive and absorptive features and an antisymmetric spectrum similar to other porphyrin triplet states. This work demonstrates that the Pchlide triplet is accessible to various cryogenic spectroscopic probes over a range of time scales and paves the way for understanding its potential role in catalysis.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/metabolismo , Análisis Espectral , Catálisis , Clorofila , Fotoquímica , Protoclorofilida/química , SolventesRESUMEN
Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654â nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Galactolípidos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fosfatidilgliceroles/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión , Clorofila/biosíntesis , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Galactolípidos/química , Expresión Génica , Cinética , Luz , Modelos Moleculares , Mutación , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Fosfatidilgliceroles/química , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Protoclorofilida/química , Protoclorofilida/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase. Pchlide has excited-state properties that allow it to initiate photochemistry in the enzyme active site, which involves reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical behavior has been investigated here using a combination of time-resolved spectroscopies and density functional theory calculations of a number of Pchlide analogues with modifications to various substituent groups. A keto group on ring E is essential for excited-state charge separation in the molecule, which is the driving force for the photoreactivity of the pigment. Vibrational "fingerprints" of specific regions of the Pchlide chromophore have been assigned, allowing identification of the modes that are crucial for excited-state chemistry in the enzyme. This work provides an understanding of the structural determinants of Pchlide that are important for harnessing light energy.
Asunto(s)
Clorofila/síntesis química , Luz , Compuestos Organometálicos/química , Protoclorofilida/química , Teoría Cuántica , Clorofila/química , Estructura Molecular , Protoclorofilida/análogos & derivadosRESUMEN
Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME-cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME-cyclase activity in the cyanobacterium SynechocystisSynechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME-cyclase step, whereas the A9G mutation reduced Ycf54 levels by â¼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by â¼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME-cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.
Asunto(s)
Proteínas Bacterianas/química , Oxigenasas/química , Subunidades de Proteína/química , Protoclorofilida/biosíntesis , Proteínas Recombinantes/química , Synechocystis/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Ciclización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , Oxigenasas/genética , Oxigenasas/metabolismo , Fotosíntesis/genética , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Protoclorofilida/química , Protoporfirinas/química , Protoporfirinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática , Synechocystis/enzimologíaRESUMEN
Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.
Asunto(s)
Luz , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/química , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Estructura Terciaria de Proteína , Protoclorofilida/química , Alineación de Secuencia , EspectrofotometríaRESUMEN
Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His6-PORA, His6-PORB and His6-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Fluorescencia , Modelos Moleculares , Complejos Multienzimáticos/química , Protoclorofilida/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálisis , Complejos Multienzimáticos/genética , Protoclorofilida/genéticaRESUMEN
The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called "reactive" intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.
Asunto(s)
Luz , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Biocatálisis , Clorofilidas/química , Clorofilidas/metabolismo , Transporte de Electrón , NADP/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Procesos Fotoquímicos , Protoclorofilida/química , Protoclorofilida/metabolismo , Espectrofotometría InfrarrojaRESUMEN
The purpose of this review is to summarize and discuss data obtained in studies on the mechanisms of the primary photophysical and photochemical reactions of protochlorophyllide photoreduction in plant materials (etiolated leaves and leaf homogenates) and in model systems. Based on the results of numerous studies, it can be stated that the reduction of active forms of the chlorophyll precursor is a multistep process comprising two or three short-lived intermediates characterized by a singlet ESR signal. The first intermediate is probably a complex with charge transfer between protochlorophyllide and the hydride ion donor NADPH. The conserved tyrosine residue Tyr193 of protochlorophyllide oxidoreductase is the donor of the second proton.
Asunto(s)
Clorofilidas/metabolismo , Protoclorofilida/metabolismo , Clorofilidas/química , Procesos Fotoquímicos , Protoclorofilida/químicaRESUMEN
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.
Asunto(s)
Bacterioclorofila A/síntesis química , Hierro/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Protoclorofilida/química , Azufre/química , Bacterioclorofila A/biosíntesis , Activación Enzimática/efectos de la radiación , Radicales Libres , Hierro/efectos de la radiación , Luz , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/efectos de la radiación , Fotosíntesis/fisiología , Fotosíntesis/efectos de la radiación , Protoclorofilida/efectos de la radiación , Especificidad por Sustrato , Azufre/efectos de la radiaciónRESUMEN
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.