ABSTRACT
The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1-3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.
Subject(s)
Chlorophyll/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Synechococcus/enzymology , Synechocystis/enzymology , Catalysis , Chlorophyll/chemistry , Molecular Structure , Photochemistry , Protochlorophyllide/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli. We demonstrate however that blue light-exposure, which is required for photodecarboxylase activity, also leads to irreversible inactivation of the enzyme, especially in the absence of palmitate substrate. Photoinactivation is attributed to formation of protein based organic radicals, which were observed by EPR spectroscopy. To suppress photoinactivation, we prepared stable and catalytically active FAP in the dark. The steady-state kinetic parameters of CvFAP (kcat: 0.31 ± 0.06 s-1 and KM: 98.8 ± 53.3 µM) for conversion of palmitic acid to pentadecane were determined using gas chromatography. Methods described here should now enable studies of the catalytic mechanism and exploitation of FAPs in biotechnology.
Subject(s)
Carboxy-Lyases/metabolism , Fatty Acids/metabolism , Biocatalysis , Carboxy-Lyases/chemistry , Escherichia coli/enzymology , Fatty Acids/chemistry , Free Radicals/chemistry , Free Radicals/metabolism , Kinetics , Photochemical ProcessesABSTRACT
Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.
Subject(s)
Chlorophyta/chemistry , Phytochrome/chemistry , Photochemical Processes , Protein Conformation , Spectrophotometry, Infrared , X-Ray DiffractionABSTRACT
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.
Subject(s)
Cyanobacteria , Oxidoreductases Acting on CH-CH Group Donors , Protochlorophyllide/chemistry , Light , Protons , Oxidoreductases Acting on CH-CH Group Donors/metabolism , PhotochemistryABSTRACT
Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Subject(s)
Bile Pigments , Photoreceptors, Microbial , Photochemistry , Biliverdine , Bacterial Proteins/metabolism , Photoreceptors, Microbial/chemistry , LightABSTRACT
CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
Subject(s)
Cold Temperature , Histidine , Ligands , Oxidation-Reduction , LightingABSTRACT
Protein dynamics are crucial for realizing the catalytic power of enzymes, but how enzymes have evolved to achieve catalysis is unknown. The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively, and is an excellent system for relating the effects of motions to catalysis. Here, we have used the temperature dependence of isotope effects and solvent viscosity measurements to analyze the dynamics coupled to the hydride and proton transfer steps in three cyanobacterial PORs and a related plant enzyme. We have related the dynamic profiles of each enzyme to their evolutionary origin. Motions coupled to light-driven hydride transfer are conserved across all POR enzymes, but those linked to thermally activated proton transfer are variable. Cyanobacterial PORs require complex and solvent-coupled dynamic networks to optimize the proton donor-acceptor distance, but evolutionary pressures appear to have minimized such networks in plant PORs. POR from Gloeobacter violaceus has features of both the cyanobacterial and plant enzymes, suggesting that the dynamic properties have been optimized during the evolution of POR. We infer that the differing trajectories in optimizing a catalytic structure are related to the stringency of the chemistry catalyzed and define a functional adaptation in which active site chemistry is protected from the dynamic effects of distal mutations that might otherwise impact negatively on enzyme catalysis.
Subject(s)
Bacterial Proteins/chemistry , Cyanobacteria/enzymology , Evolution, Molecular , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Plant Proteins/chemistry , Plants/enzymology , Protons , Bacterial Proteins/metabolism , Ion Transport/physiology , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Plant Proteins/metabolismABSTRACT
Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.
Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Bacterial Proteins/metabolism , Cobamides/chemistry , Cobamides/genetics , Cobamides/metabolism , DNA/metabolism , Phosphothreonine/analogs & derivatives , Thermus thermophilus/genetics , Thermus thermophilus/metabolism , Vitamin B 12/metabolismABSTRACT
We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,ß-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.
Subject(s)
Enterobacter cloacae/enzymology , Mutagenesis, Site-Directed , Oxidoreductases/genetics , Oxidoreductases/metabolism , Shewanella/enzymology , Aldehydes/metabolism , Alkenes/metabolism , Crystallography, X-Ray , Enterobacter cloacae/chemistry , Enterobacter cloacae/genetics , Models, Molecular , Oxidoreductases/chemistry , Oximes/metabolism , Phenols/metabolism , Protein Binding , Shewanella/chemistry , Shewanella/genetics , Stereoisomerism , Substrate SpecificityABSTRACT
Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.
ABSTRACT
We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.
ABSTRACT
H(+) but not H(-): The reduction reaction of protochlorophyllide catalyzed by protochlorophyllide oxidoreductase features solvent-slaved motions that control the proton- but not the hydride-tunneling mechanism. These motions imply a long-range dynamic network from the solvent to the enzyme active site that facilitate proton transfer (see picture, left). Motions for hydride transfer are more localized and are not slaved by the solvent (see picture, right).
Subject(s)
Hydrogen/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Protons , Solvents/chemistry , Cyanobacteria/enzymology , Enzyme Activation/radiation effects , Light , Molecular Structure , Photochemical Processes , ViscosityABSTRACT
Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood-brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer's disease, and Parkinson's disease.
Subject(s)
Brain/drug effects , Kynurenine 3-Monooxygenase/metabolism , Neurodegenerative Diseases/metabolism , Animals , Blood-Brain Barrier , Brain/metabolism , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/metabolism , Kynurenine 3-Monooxygenase/antagonists & inhibitors , Mice , Neurodegenerative Diseases/enzymologyABSTRACT
The light-driven enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary enzyme-substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the enzyme active site. This is followed by the binding of the Pchlide substrate and subsequent substrate-induced conformational changes within the enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein.
Subject(s)
Light , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Catalysis , Fluorescence Resonance Energy Transfer , Kinetics , NADP/metabolism , Protein Binding , Protein Conformation , Spectroscopy, Fourier Transform Infrared , Substrate SpecificityABSTRACT
Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel 'head-to-head' arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Light , Phytochrome/chemistry , Phytochrome/metabolism , Protein Kinases/chemistry , Protein Kinases/metabolism , Signal Transduction/radiation effects , Synechocystis/radiation effects , Absorption/radiation effects , Color , Electron Spin Resonance Spectroscopy , Isomerism , Kinetics , Movement/radiation effects , Photoreceptors, Microbial , Phycobilins/chemistry , Phycobilins/metabolism , Phycocyanin/chemistry , Phycocyanin/metabolism , Protein Conformation/radiation effects , Solvents/chemistry , Synechocystis/cytology , Synechocystis/metabolism , Time FactorsABSTRACT
Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome--1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.
Subject(s)
Circadian Rhythm/physiology , Cryptochromes/metabolism , Flavins/metabolism , Metamorphosis, Biological/physiology , Signal Transduction/physiology , Animals , Animals, Genetically Modified , Cryptochromes/isolation & purification , DNA Primers/genetics , Darkness , Drosophila , Gene Expression Profiling , Humans , Microarray Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In chlorophyll biosynthesis, the light-activated enzyme protochlorophyllide oxidoreductase catalyzes trans addition of hydrogen across the C-17-C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). This unique light-driven reaction plays a key role in the assembly of the photosynthetic apparatus, but despite its biological importance, the mechanism of light-activated catalysis is unknown. In this study, we show that Pchlide reduction occurs by dynamically coupled nuclear quantum tunneling of a hydride anion followed by a proton on the microsecond time scale in the Pchlide excited and ground states, respectively. We demonstrate the need for fast dynamic searches to form degenerate "tunneling-ready" configurations within the lifetime of the Pchlide excited state from which hydride transfer occurs. Moreover, we have found a breakpoint at -27 degrees C in the temperature dependence of the hydride transfer rate, which suggests that motions/vibrations that are important for promoting light-activated hydride tunneling are quenched below -27 degrees C. We observed no such breakpoint for the proton-tunneling reaction, indicating a reliance on different promoting modes for this reaction in the enzyme-substrate complex. Our studies indicate that the overall photoreduction of Pchlide is endothermic and that rapid dynamic searches are required to form distinct tunneling-ready configurations within the lifetime of the photoexcited state. Consequently, we have established the first important link between photochemical and nuclear quantum tunneling reactions, linked to protein dynamics, in a biologically significant system.
Subject(s)
Bacterial Proteins/chemistry , Cyanobacteria/enzymology , Light , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Protochlorophyllide/chemistry , Protons , Bacterial Proteins/metabolism , Oxidation-Reduction , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Photochemistry , Protochlorophyllide/metabolismABSTRACT
The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.