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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. [ABSTRACT FROM AUTHOR]

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]

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. CarH is a bacterial B12-binding photoreceptor involved in transcriptional regulation. Here, the authors provide insights into B12 dynamics and associated cobalt redox changes following light activation. These demonstrate the CarH response integrates light and oxygen sensing. [ABSTRACT FROM AUTHOR]

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides (1). Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle (2, 3). Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria (4, 5), with substrates including polychlorinated biphenyls or dioxins (6, 7). Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies (8, 12). Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies (13), we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis., Cobalamin consists of a cobalt ion coordinated by a tetrapyrrole-derived macrocycle and readily forms a weak organometallic C-Co(III) bond (14). This bond can be homolytically or heterolytically cleaved and the [...]

Access to new non‐canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored—despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non‐natural methionines through the development of a photochemical method for the radical α‐C−H functionalization of sulfides with alkenes, in water, using inexpensive and commercially‐available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two‐step synthesis of novel methionine analogues—by radical addition to unsaturated amino acid derivatives—and the chemoselective modification of peptide side‐chains to yield non‐natural methionine residues within small peptides. The mechanism of the bio‐inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies. [ABSTRACT FROM AUTHOR]