Photocobilins integrate B12 and bilin photochemistry for enzyme control.

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.

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

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.

Direct Comparison between Förster Resonance Energy Transfer and Light-Induced Triplet-Triplet Electron Resonance Spectroscopy.

To carry out reliable and comprehensive structural investigations, the exploitation of different complementary techniques is required. Here, we report that dual triplet-spin/fluorescent labels enable the first parallel distance measurements by electron spin resonance (ESR) and Förster resonance energy transfer (FRET) on exactly the same molecules with orthogonal chromophores, allowing for direct comparison. An improved light-induced triplet-triplet electron resonance method with 2-color excitation is used, improving the signal-to-noise ratio of the data and yielding a distance distribution that provides greater insight than the single distance resulting from FRET.

Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

Redox driven B12-ligand switch drives CarH photoresponse.

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.

How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

Vanadium haloperoxidases (VHPOs) are unique enzymes in biology that catalyze a challenging halogen transfer reaction and convert a strong aromatic C-H bond into C-X (X = Cl, Br, I) with the use of a vanadium cofactor and H2O2. The VHPO catalytic cycle starts with the conversion of hydrogen peroxide and halide (X = Cl, Br, I) into hypohalide on the vanadate cofactor, and the hypohalide subsequently reacts with a substrate. However, it is unclear whether the hypohalide is released from the enzyme or otherwise trapped within the enzyme structure for the halogenation of organic substrates. A substrate-binding pocket has never been identified for the VHPO enzyme, which questions the role of the protein in the overall reaction mechanism. Probing its role in the halogenation of small molecules will enable further engineering of the enzyme and expand its substrate scope and selectivity further for use in biotechnological applications as an environmentally benign alternative to current organic chemistry synthesis. Using a combined experimental and computational approach, we elucidate the role of the vanadium haloperoxidase protein in substrate halogenation. Activity studies show that binding of the substrate to the enzyme is essential for the reaction of the hypohalide with substrate. Stopped-flow measurements demonstrate that the rate-determining step is not dependent on substrate binding but partially on hypohalide formation. Using a combination of molecular mechanics (MM) and molecular dynamics (MD) simulations, the substrate binding area in the protein is identified and even though the selected substrates (methylphenylindole and 2-phenylindole) have limited hydrogen-bonding abilities, they are found to bind relatively strongly and remain stable in a binding tunnel. A subsequent analysis of the MD snapshots characterizes two small tunnels leading from the vanadate active site to the surface that could fit small molecules such as hypohalide, halide, and hydrogen peroxide. Density functional theory studies using electric field effects show that a polarized environment in a specific direction can substantially lower barriers for halogen transfer. A further analysis of the protein structure indeed shows a large dipole orientation in the substrate-binding pocket that could enable halogen transfer through an applied local electric field. These findings highlight the importance of the enzyme in catalyzing substrate halogenation by providing an optimal environment to lower the energy barrier for this challenging aromatic halide insertion reaction.

Photoinduced Electron Transfer from a 1,4,5,6-Tetrahydro Nicotinamide Adenine Dinucleotide (Phosphate) Analogue to Oxidized Flavin in an Ene-Reductase Flavoenzyme.

Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H4 to FMN, generating a NAD(P)H4 cation radical and anionic FMN semiquinone. This electron transfer occurs in ∼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over ∼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.

Catalysis by Nature's photoenzymes.

Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biological photocatalysis.

A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.

Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron-electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis.

The photochemical reaction catalyzed by enzyme protochlorophyllide oxidoreductase (POR), a rare example of a photoactivated enzyme, is a crucial step during chlorophyll biosynthesis and involves the fastest known biological hydride transfer. Structures of the enzyme with bound substrate protochlorophyllide (PChlide) and coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) have recently been published, opening up the possibility of using computational approaches to provide a comprehensive understanding of the excited state chemistry. Herein, we propose a complete mechanism for the photochemistry between PChlide and NADPH based on density functional theory (DFT) and time-dependent DFT calculations that is consistent with recent experimental data. In this multi-step mechanism, photoexcitation of PChlide leads to electron transfer from NADPH to PChlide, which in turn facilitates hydrogen atom transfer by weakening the breaking C-H bond. This work rationalizes how photoexcitation facilitates hydride transfer in POR and has more general implications for biological hydride transfer reactions.

An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

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.

A guide to time-resolved structural analysis of light-activated proteins.

Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.

A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Design and fabrication of recombinant reflectin-based multilayer reflectors: bio-design engineering and photoisomerism induced wavelength modulation.

The remarkable camouflage capabilities of cephalopods have inspired many to develop dynamic optical materials which exploit certain design principles and/or material properties from cephalopod dermal cells. Here, the angle-dependent optical properties of various single-layer reflectin thin-films on Si wafers are characterized within the UV-Vis-NIR regions. Following this, initial efforts to design, fabricate, and optically characterize a bio-inspired reflectin-based multilayer reflector is described, which was found to conserve the optical properties of single layer films but exhibit reduced angle-dependent visible reflectivity. Finally, we report the integration of phytochrome visible light-induced isomerism into reflectin-based films, which was found to subtly modulate reflectin thin-film reflectivity.

Photocatalysis as the 'master switch' of photomorphogenesis in early plant development.

Enzymatic photocatalysis is seldom used in biology. Photocatalysis by light-dependent protochlorophyllide oxidoreductase (LPOR)-one of only a few natural light-dependent enzymes-is an exception, and is responsible for the conversion of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis. Photocatalysis by LPOR not only regulates the biosynthesis of the most abundant pigment on Earth but it is also a 'master switch' in photomorphogenesis in early plant development. Following illumination, LPOR promotes chlorophyll production, plastid membranes are transformed and the photosynthetic apparatus is established. Given these remarkable, light-induced pigment and morphological changes, the LPOR-catalysed reaction has been extensively studied from catalytic, physiological and plant development perspectives, highlighting vital, and multiple, cellular roles of this intriguing enzyme. Here, we offer a perspective in which the link between LPOR photocatalysis and plant photomorphogenesis is explored. Notable breakthroughs in LPOR structural biology have uncovered the structural-mechanistic basis of photocatalysis. These studies have clarified how photon absorption by the pigment protochlorophyllide-bound in a ternary LPOR-protochlorophyllide-NADPH complex-triggers photocatalysis and a cascade of complex molecular and cellular events that lead to plant morphological changes. Photocatalysis is therefore the master switch responsible for early-stage plant development and ultimately life on Earth.

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2 O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2 O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2 O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2 O2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H2 O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2 O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2 O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.