Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.

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 lightdriven 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°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°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.Hydrogen transfer reactions are fundamental chemical processes that are essential for almost all biological reactions. H-transfer by tunneling is an important feature of these reactions in enzymes (1-3), but mechanistic understanding of how protein motions (from the millisecond to sub-picosecond time domain) facilitate the H-tunneling reactions remains elusive (4 -6). A major limitation has been the inability to synchronously trigger catalysis on ultrafast time scales for the majority of enzymes that require mixing strategies to initiate the reaction. However, by using the light-activated enzyme, protochlorophyllide oxidoreductase (POR 4 ; EC 1.3.1.33) (7), we have triggered two enzymatic H-transfer reactions using a single pulse of light, and we show these reactions occur sequentially by quantum tunneling in a pre-formed enzyme-substrate complex. This has provided a unique opportunity to analyze these reactions at physiological and cryogenic temperatures, on very fast time scales, that are experimentally inaccessible with other enzyme systems.POR catalyzes the trans addition of hydrogen across the C-17-C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide) to produce chlorophyllide (Chlide) (7), a unique light-driven reaction in the synthesis of the most abundant pigment on earth, which plays a key role in the assembly of the photosynthetic apparatus (8, 9). In addition to POR, non...

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

No abstract

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a lightdependent step in chlorophyll biosynthesis that is essential to photosynthesis and ultimately all life on Earth. 1-3 POR, which is one of three known light-dependent enzymes, 4,5 catalyzes reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, a structural basis for POR photocatalysis has remained elusive. Here, we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex have identified 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 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. As the light-driven step in the chlorophyll biosynthetic pathway (Fig. 1), the POR reaction acts as the trigger for the germination of seedlings =in plants and provokes a marked change in the morphological development of the plant. 2,3 Given this crucial biological role, POR has been the focus of numerous mechanistic and biophysical investigations. A combination of time-resolved (at the femtosecond-to-second scale) and cryogenic spectroscopy methods have provided some understanding of the mechanism of POR photocatalysis in a range of photosynthetic organisms, including cyanobacteria and plants. Picosecond excited-state dynamics in the protochlorophyllide (Pchlide) molecule are thought to result in excited state interactions between the substrate and active-site residues that are necessary to trigger the subsequent reaction chemistry. 6-12 This involves sequential transfer of a hydride equivalent from NADPH and a proton transfer from either an active site residue or solvent. Proton transfer is reliant on solvent dynamics and an implied network of extended protein motions that occur on the microsecond timescale. 13-17 Hydride transfer from NADPH is not concerted, but occurs in a stepwise manner that involves

Strategy for bio-alkane gas (propane and butane) production through the conversion of waste volatile fatty acids by bacterial cultures.

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH. As POR is light-dependent, catalysis can be initiated by illumination of the enzyme-substrate complex at low temperatures, making it an attractive model for studying aspects of biological proton and hydride transfers. The early stages in the photoreduction, involving Pchlide binding and an initial photochemical reaction, have been studied in vitro by using low-temperature fluorescence and absorbance measurements. Formation of the ternary POR-NADPHPchlide complex produces red shifts in the fluorescence and absorbance maxima of Pchlide, allowing the dissociation constant for Pchlide binding to be measured. We demonstrate that the product of an initial photochemical reaction, which can occur below 200 K, is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A696) that is suggested to represent an ion radical complex. The temperature dependence of the rate of A696 formation has allowed the activation energy for the photochemical step to be calculated and has shown that POR catalysis can proceed at much lower temperatures than previously thought. Calculations of differences in free energy between various reaction intermediates have been calculated; these, together with the quantum efficiency for Pchlide conversion, suggest a quantitative model for the thermodynamics of the light-driven step of Pchlide reduction.

Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the singlet spin state occurs. As revealed by steady- and transient-state kinetics the oxygen-dependent steps are rate-limiting, whereas Trp160 and His251 are essential residues for catalysis and contribute to substrate positioning and activation, respectively. Computational modeling further confirms the experimental observations and rationalizes the electron transfer pathways, and the effect of substrate and substrate binding pocket residues. Finally, we make a direct comparison with iron-based dioxygenases and explain the mechanistic and electronic differences with cofactor-free dioxygenases. Our multidisciplinary study confirms that the oxygenation reaction can take place in absence of any cofactor by a unique mechanism in which the specially designed fit-for-purpose active-site architecture modulates substrate reactivity toward oxygen.