Novel Types of Phyllobilins in a Fern - Molecular Reporters of the Evolution of Chlorophyll Breakdown in the Paleozoic Era.

Breakdown of chlorophyll (Chl), as studied in angiosperms, follows the pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway, furnishing linear tetrapyrroles, named phyllobilins (PBs). In an investigation with fern leaves we have discovered iso-phyllobilanones (iPBs) with an intriguingly rearranged and oxidized carbon skeleton. We report here a key second group of iPBs from the fern and on their structure analysis. Previously, these additional Chl-catabolites escaped their characterization, since they exist in aqueous media as mixtures of equilibrating isomers. However, their chemical dehydration furnished stable iPB-derivatives that allowed the delineation of the enigmatic structures and chemistry of the original natural catabolites. The structures of all fern-iPBs reflect the early core steps of a PaO/PB-type pathway and the PB-to-iPB carbon skeleton rearrangement. A striking further degradative chemical ring-cleavage was observed, proposed to consume singlet molecular oxygen (1O2). Hence, Chl-catabolites may play a novel active role in detoxifying cellular 1O2. The critical deviations from the PaO/PB pathway, found in the fern, reflect evolutionary developments of Chl-breakdown in the green plants in the Paleozoic era.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Cryogenic Ion Vibrational Predissociation (CIVP) Spectroscopy of Aryl Cobinamides in the Gas Phase: How Good Are the Calculations for Vitamin B12 Derivatives?

Aryl corrins represent a novel class of designed B12 derivatives with biological properties of "antivitamins B12". In our previous study, we experimentally determined bond strength in a series of aryl-corrins by the threshold collision-induced dissociation experiments (T-CID) and compared the measured bond dissociation energies (BDEs) with those calculated with density functional theory (DFT). We found that the BDEs are modulated by the side chains around the periphery of the corrin unit. Given that aryl cobinamides have many side chains that increase their conformational space and that the question of a specific structure, measured in the gas phase, was important for further evaluation of our T-CID experiment, we proceeded to analyze structural properties of aryl cobinamides using cryogenic ion vibrational predissociation (CIVP) spectroscopy, static DFT, and Born-Oppenheimer molecular dynamic (BOMD) simulations. We found that none of the examined DFT models could reproduce the CIVP spectra convincingly; both "static" DFT calculations and "dynamic" BOMD simulations provide a surprisingly poor representation of the vibrational spectra, specifically of the number, position, and intensity of bands assigned to hydrogen-bonded versus non-hydrogen-bonded NH and OH moieties. We conclude that, for a flexible molecule with ca. 150 atoms, more accurate approaches are needed before definitive conclusions about computed properties, specifically the structure of the ground-state conformer, may be made.

A Chlorophyll-Derived Phylloxanthobilin Is a Potent Antioxidant That Modulates Immunometabolism in Human PBMC.

Phyllobilins are natural products derived from the degradation of chlorophyll, which proceeds via a common and strictly controlled pathway in higher plants. The resulting tetrapyrrolic catabolites-the phyllobilins-are ubiquitous in nature; despite their high abundance, there is still a lack of knowledge about their physiological properties. Phyllobilins are part of human nutrition and were shown to be potent antioxidants accounting with interesting physiological properties. Three different naturally occurring types of phyllobilins-a phylloleucobilin, a dioxobilin-type phylloleucobilin and a phylloxanthobilin (PxB)-were compared regarding potential antioxidative properties in a cell-free and in a cell-based antioxidant activity test system, demonstrating the strongest effect for the PxB. Moreover, the PxB was investigated for its capacity to interfere with immunoregulatory metabolic pathways of tryptophan breakdown in human blood peripheral mononuclear cells. A dose-dependent inhibition of tryptophan catabolism to kynurenine was observed, suggesting a suppressive effect on pathways of cellular immune activation. Although the exact mechanisms of immunomodulatory effects are yet unknown, these prominent bioactivities point towards health-relevant effects, which warrant further mechanistic investigations and the assessment of the in vivo extrapolatability of results. Thus, phyllobilins are a still surprisingly unexplored family of natural products that merit further investigation.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

Antivitamins B12: Synthesis and application as inhibitory ligand of the B12-tailoring enzyme CblC.

Antivitamins B12 are non-natural corrinoids that have been designed to counteract the metabolic effects of vitamin B12 and related cobalamins (Cbls) in humans and other mammals. A basic structure- and reactivity-based concept typifies antivitamins B12 as close structural mimics of vitamin B12 that are not transformed by the cellular metabolism into organometallic B12-cofactors. Antivitamins B12 have the correct structure for efficient take-up and transport via the natural mammalian pathway for cobalamin assimilation. Thus they can be delivered to every cell in the body, where they are proposed to target and inhibit the Cbl tailoring enzyme CblC. Antivitamins B12 may be specifically inert Cbls or isostructural Cbl-analogues that carry a metal centre other than a cobalt-ion. The syntheses of two antivitamins B12 are detailed here, as are biochemical and crystallographic studies that provide insights into the crucial binding interactions of Cbl-based antivitamins B12 with the human B12-tailoring enzyme CblC. This key enzyme binds genuine antivitamins B12 as inert substrate mimics and enzyme inhibitors, effectively repressing the metabolic generation of the B12-cofactors. Hence, antivitamins B12 induce the diagnostic symptoms of (functional) B12-deficiency, as observed in healthy laboratory mice.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012-fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

An evergreen mind and a heart for the colors of fall.

With the finest biochemical and molecular approaches, convincing explorative strategies, and long-term vision, Stefan Hörtensteiner succeeded in elucidating the biochemical pathway responsible for chlorophyll degradation. After having contributed to the identification of key chlorophyll degradation products in the course of the past 25 years, he gradually identified and characterized most of the crucial players in the PAO/phyllobilin degradation pathway of chlorophyll. He was one of the brightest plant biochemists of his generation, and his work opened doors to a better understanding of plant senescence, tetrapyrrole homeostasis, and their complex regulation. He sadly passed away on 5 December 2020, aged 57.

Surprising Homolytic Gas Phase Co-C Bond Dissociation Energies of Organometallic Aryl-Cobinamides Reveal Notable Non-Bonded Intramolecular Interactions.

Aryl-cobalamins are a new class of organometallic structural mimics of vitamin B12 designed as potential 'antivitamins B12 '. Here, the first cationic aryl-cobinamides are described, which were synthesized using the newly developed diaryl-iodonium method. The aryl-cobinamides were obtained as pairs of organometallic coordination isomers, the stereo-structure of which was unambiguously assigned based on homo- and heteronuclear NMR spectra. The availability of isomers with axial attachment of the aryl group, either at the 'beta' or at the 'alpha' face of the cobalt-center allowed for an unprecedented comparison of the organometallic reactivity of such pairs. The homolytic gas-phase bond dissociation energies (BDEs) of the coordination-isomeric phenyl- and 4-ethylphenyl-cobinamides were determined by ESI-MS threshold CID experiments, furnishing (Co-C sp 2 )-BDEs of 38.4 and 40.6 kcal mol-1 , respectively, for the two ß-isomers, and the larger BDEs of 46.6 and 43.8 kcal mol-1 for the corresponding α-isomers. Surprisingly, the observed (Co-C sp 2 )-BDEs of the Coß -aryl-cobinamides were smaller than the (Co-C sp 3 )-BDE of Coß -methyl-cobinamide. DFT studies and the magnitudes of the experimental (Co-C sp 2 )-BDEs revealed relevant contributions of non-bonded interactions in aryl-cobinamides, notably steric strain between the aryl and the cobalt-corrin moieties and non-bonded interactions with and among the peripheral sidechains.

Antivitamins B12 -Some Inaugural Milestones.

The recently delineated structure- and reactivity-based concept of antivitamins B12 has begun to bear fruit by the generation, and study, of a range of such B12 -dummies, either vitamin B12 -derived, or transition metal analogues that also represent potential antivitamins B12 or specific B12 -antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B12 -chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B12 have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B12 -deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B12 to induce functional B12 -deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.

Replacement of the Cobalt Center of Vitamin B12 by Nickel: Nibalamin and Nibyric Acid Prepared from Metal-Free B12 †Ligands Hydrogenobalamin and Hydrogenobyric Acid.

The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transition-metal analogue of vitamin B12 . Described here is Nibl, synthesized by incorporation of a NiII ion into the metal-free B12  ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiII  corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal-free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X-ray analysis of Niby shows the structural adaptation of the corrin ligand to NiII ions and the coordination behavior of NiII . The diamagnetic Niby and Nibl, and corresponding isoelectronic CoI corrins, were deduced to be isostructural. Nibl is a structural mimic of four-coordinate base-off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase.

Ultrafast Excited State Dynamics and Fluorescence from Vitamin B12 and Organometallic [Co]-C≡C-R Cobalamins.

Cobalamins are cobalt-centered cyclic tetrapyrrole ring-based molecules that provide cofactors for exceptional biological processes and possess unique and synthetically tunable photochemistry. Typical cobalamins are characterized by a visible absorption spectrum consisting of peaks labeled α, ß, and sh. The physical basis of these peaks as having electronic origin or as a vibronic progression is ambiguous despite much investigation. Here, for the first time, cobalamin fluorescence is identified in several derivatives. The fluorescence lifetime is ca. 100-200 fs with quantum yields on the order of 10-6-10-5 because of rapid population of "dark" excited states. The results are compared with the fluorescent analogue with zinc replacing the cobalt in the corrin ring. Analysis of the breadth of the emission spectrum provides evidence that a vibrational progression in a single excited electronic state makes the dominant contribution to the visible absorption band.

Phyllobilins from Senescence-Associated Chlorophyll Breakdown in the Leaves of Basil (Ocimum basilicum) Show Increased Abundance upon Herbivore Attack.

In view of the common use of the herb basil (Ocimum basilicum) in nutrition and in phytomedicine, the contents of its leaves are of obvious interest. In extracts of fresh yellowish-green basil leaves, phyllobilins (PBs), which are bilin-type catabolites of chlorophyll (Chl), were detected using high-performance liquid chromatography (HPLC). Two such PBs, provisionally named Ob-nonfluorescent chlorophyll catabolite (NCC)-40 and Ob-YCC-45, exhibited previously unknown structures that were delineated by a thorough spectroscopic characterization. When basil leaves were infested with aphids or thrips or underwent fungal infections, areas with chlorosis were observed. HPLC analyses of the infested parts of leaves compared to those of the healthy parts showed a significant accumulation of PBs in the infested areas, demonstrating that the senescence-associated pheophorbide a oxygenase/phyllobilin (PAO/PB) pathway is activated by herbivore feeding and fungal infection.

The intermolecular anthracene-transfer in a regiospecific antipodal C60 difunctionalization.

Ever since the discovery of fullerenes, their mono- and multi-functionalization by exohedral addition chemistry has been a fundamental topic. A few years ago, a topochemically controlled regiospecific difunctionalization of C60 fullerene by anthracene in the solid state was discovered. In the present work, we analyse the mechanism of this unique reaction, where an anthracene molecule is transferred from one C60 mono-adduct to another one, under exclusive formation of equal amounts of C60 and of the difficult to make, highly useful, antipodal C60 bis-adduct. Our herein disclosed dispersion corrected DFT studies show the anthracene transfer to take place in a synchronous retro Diels-Alder/Diels-Alder reaction: an anthracene molecule dissociates from one fullerene under formation of an intermediate, while undergoing stabilizing interactions with both neighbouring fullerene molecules, facilitating the reaction kinetically. In the intermediate, a planar anthracene molecule is sandwiched between two neighbouring fullerenes and forms equally strong 'double-decker' type π-π stacking interactions with both of these fullerenes. Analysis with the distortion interaction model shows that the anthracene unit of the intermediate is almost planar with minimal distortion. This analysis highlights the existence of simultaneous noncovalent interactions engaging both faces of a planar polyunsaturated ring and two convex fullerene surfaces in an unprecedented 'inverted sandwich' structure. Hence, it sheds light on new strategies to design functional fullerene based materials.

The red chlorophyll catabolite (RCC) is an inefficient sensitizer of singlet oxygen - photochemical studies of the methyl ester of RCC.

The red chlorophyll catabolite (RCC) is a proposed cryptic intermediate of chlorophyll (Chl) breakdown in higher plants. Its accumulation in higher plants is believed to be metabolically suppressed, as RCC is commonly suspected to efficiently sensitize for the formation of the cell poison singlet oxygen (1O2). We report here a study on luminescence of the methyl ester of RCC (Me-RCC) and of its capacity to generate 1O2 in ethanolic solution. A solution of Me-RCC fluoresces at room temperature with a maximum near 670 nm and features a fluorescence spectrum with pronounced vibrational spacing at 77 K. As shown here, sensitization of the generation of 1O2 by Me-RCC in an oxygen-saturated solution in hexadeutero-ethanol occurs with a maximal quantum yield of only about 0.015. This low quantum yield suggests that the specific catabolic suppression of the accumulation of RCC during Chl breakdown is not primarily a countermeasure against the formation of 1O2 by RCC in the plant, but has other crucial reasons mainly.

Exceptional Photochemical Stability of the Co-C Bond of Alkynyl Cobalamins, Potential Antivitamins B12 and Core Elements of B12-Based Biological Vectors.

Alkynylcorrinoids are a class of organometallic B12 derivatives, recently rediscovered for use as antivitamins B12 and as core components of B12-based biological vectors. They feature exceptional photochemical and thermal stability of their characteristic extra-short Co-C bond. We describe here the synthesis and structure of 3-hydroxypropynylcobalamin (HOPryCbl) and photochemical experiments with HOPryCbl, as well as of the related alkynylcobalamins: phenylethynylcobalamin and difluoro-phenylethynylcobalamin. Ultrafast spectroscopic studies of the excited state dynamics and mechanism for ground state recovery demonstrate that the Co-C bond of alkynylcobalamins is stable, with the Co-N bond and ring deformations mediating internal conversion and ground state recovery within 100 ps. These studies provide insights required for the rational design of photostable or photolabile B12-based cellular vectors.

Using sliding mode observers to estimate BtuB concentration from measured vitamin B12 concentration.

A simple model for the B12-riboswitch regulatory network in Escherichia coli is first described and the same analysis is applied when changing the strain to Salmonella enterica. Model validation is undertaken by linking the dynamics of the riboswitch model to bacterial growth and comparing the results obtained with in vivo experimental measurements. Measurements of bacterial growth are relatively straightforward to obtain experimentally, but experimental measurements relating to the operation of the riboswitch are more difficult. Using the validated model, sliding mode observer design methods are used to estimate BtuB given measurements of the concentration of vitamin B12. The sliding mode approach is selected because of its inherent robustness properties as well as for the ease of implementation. Validation of the estimates of BtuB produced by the observer is undertaken by comparing the BtuB and vitamin B12 concentrations estimated from the observer with green fluorescent protein production and the concentration of vitamin B12 obtained experimentally. These experimental results also provide further validation of the underpinning mathematical model. The results establish that using a sliding mode observer as a soft sensor is a useful approach to explore the operation of a vitamin B12 riboswitch given measurements of the concentration of vitamin B12.

Antivitamins B12 in a Microdrop: The Excited-State Structure of a Precious Sample Using Transient Polarized X-ray Absorption Near-Edge Structure.

Polarized transient X-ray absorption near-edge structure (XANES) was used to probe the excited-state structure of a photostable B12 antivitamin (Coß-2-(2,4-difluorophenyl)-ethynylcobalamin, F2PhEtyCbl). A drop-on-demand delivery system synchronized to the LCLS X-ray free electron laser pulses was implemented and used to measure the XANES difference spectrum 12 ps following excitation, exposing only ∼45 µL of sample. Unlike cyanocobalamin (CNCbl), where the Co-C bond expands 15-20%, the excited state of F2PhEtyCbl is characterized by little change in the Co-C bond, suggesting that the acetylide linkage raises the barrier for expansion of the Co-C bond. In contrast, the lower axial Co-NDMB bond is elongated in the excited state of F2PhEtyCbl by ca. 10% or more, comparable to the 10% elongation observed for Co-NDMB in CNCbl.