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Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts. [ABSTRACT FROM AUTHOR]

While it is well established that thermally-activated quantum mechanical tunnelling of light particles (electrons and light atoms, typically hydrogen) plays a role in many enzyme-catalysed reactions, there are few definitive experimental signatures of atomic tunnelling and no clear methods of directly estimating the relative tunnelling contribution from typical experimental data. As most enzyme reactions involve the binding/capture of freely diffusing substrate(s), reactions are typically initiated by mixing and experimental conditions must then be compatible with liquid water (the solvent). This precludes the classic test of tunnelling: the observation of temperature-independent rate constants at cryogenic temperatures. Instead, H-tunnelling is usually inferred from kinetic isotope effects that are larger than the semiclassical limit. Often, the temperature dependence of the reaction is also measured over the experimentally accessible range (∼278–313 K for mesophilic enzymes), with resulting data analysed and interpreted using variations of Arrhenius, Eyring or Marcus theory. The apparent Arrhenius and Eyring activation parameters allow some quantitative comparison of different reactions, but do not directly provide any information about tunnelling, while the validity of parameters derived from non-adiabatic models such as Marcus theory are questionable due to the partially adiabatic nature of these reactions. Here, we use the correlation found between apparent activation enthalpy and entropy across several series of enzyme variants and tunnelling contributions determined using computational chemistry in an attempt to question and define new signatures of hydrogen tunnelling, which can be used to interpret typical experimental kinetic data measured for enzyme-catalysed reactions. [ABSTRACT FROM AUTHOR]

The development of graphene–polymer nanocomposite materials has been hindered by issues such as poor colloidal stability of graphene in liquid media, weak interactions between graphene and the host polymers as well as the lack of scalable and economical graphene synthesis routes. Chlorosulfonic acid (CSA) can spontaneously disperse graphene without the need for mechanical agitation, chemical functionalisation or surfactant stabilisation,1 however is incompatible with most polymers and organic materials. Here, we demonstrate how poly(p-phenylene terephthalamide) (PPTA) – the polymer which constitutes Kevlar – can be co-processed with graphene in CSA and wet-spun into nanocomposite fibres with minimal aggregation of graphene. [ABSTRACT FROM AUTHOR]

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (∼30–40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants. [ABSTRACT FROM AUTHOR]

Distinct mechanisms have been proposed for the biological dehalogenation catalyzed by cobalamin-dependent enzymes, with two recent crystallographic studies suggesting different mechanisms based on the observed interaction between the organohalide substrate and cobalamin. In one case, involving an aromatic dibromide substrate in NpRdhA, a novel CoII–Br interaction was observed using EPR, suggesting a mechanism involving a [Co…X…R] adduct. However, in the case of trichloroethylene in PceA, a significantly longer Co–Cl distance was observed in X-ray crystal structures, suggesting a dissociative electron transfer mechanism. Subsequent DFT models of these reactions have not reproduced these differences in binding modes. Here, we have performed molecular docking and DFT calculations to investigate and compare the interaction between different organohalides and cobalamin in both NpRdhA and PceA. In each case, despite differences in binding in the CoII state, the reaction likely proceeds via formation of a [Co…X…R] adduct in the CoI state that weakens the breaking carbon–halide bond, suggesting this could be a general mechanism for cobalamin-dependent dehalogenation. [ABSTRACT FROM AUTHOR]

Enzyme-catalysed H-transfer reactions are ubiquitous, yet fundamental details of these reactions remain unresolved. In this perspective, we discuss the roles of nuclear quantum tunnelling and (compressive) dynamics during these reactions. Evidence for the coupling of specific substrate and/or protein vibrations to the chemical coordinate is considered and a case is made for the combination of multiple experimental and computational/theoretical approaches when studying these reactions. [ABSTRACT FROM AUTHOR]

Gd4O2S:Yb:Tm rare-earth upconversion phosphors have been utilised to monitor the redox behaviour of flavin mononucleotide and report on the turnover of a flavo-protein, (pentaerythritol tetranitrate reductase). The presence of two bands separated by over 300 nm in the UCP emission spectra allows ratiometric signalling of these processes with high sensitivity. [ABSTRACT FROM AUTHOR]

A combination of the temperature- and pressure-dependencies of the kinetic isotope effect on the proton coupled electron transfer during ascorbate oxidation by ferricyanide suggests that this reference reaction may exploit vibrationally assisted quantum tunnelling of the transferred proton. [ABSTRACT FROM AUTHOR]

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B 12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B 12 -dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B 12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B 12 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 B 12 -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 B 12 . When B 12 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 B 12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B 12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B 12 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 B 12 -dependent photoreceptors as light-activated tools for biomedical applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho -position using pressurized CO 2 (∼30-40 bar) catalyzed by ortho -benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

Gd4O2S:Yb:Tm rare-earth upconversion phosphors have been utilised to monitor the redox behaviour of flavin mononucleotide and report on the turnover of a flavo-protein, (pentaerythritol tetranitrate reductase). The presence of two bands separated by over 300 nm in the UCP emission spectra allows ratiometric signalling of these processes with high sensitivity.