Ground-State Electron Transfer as an Initiation Mechanism for Biocatalytic C-C Bond Forming Reactions.

The development of non-natural reaction mechanisms is an attractive strategy for expanding the synthetic capabilities of substrate promiscuous enzymes. Here, we report an "ene"-reductase catalyzed asymmetric hydroalkylation of olefins using α-bromoketones as radical precursors. Radical initiation occurs via ground-state electron transfer from the flavin cofactor located within the enzyme active site, an underrepresented mechanism in flavin biocatalysis. Four rounds of site saturation mutagenesis were used to access a variant of the "ene"-reductase nicotinamide-dependent cyclohexanone reductase (NCR) from Zymomonas mobiles capable of catalyzing a cyclization to furnish ß-chiral cyclopentanones with high levels of enantioselectivity. Additionally, wild-type NCR can catalyze intermolecular couplings with precise stereochemical control over the radical termination step. This report highlights the utility for ground-state electron transfers to enable non-natural biocatalytic C-C bond forming reactions.

Photoenzymatic Reductions Enabled by Direct Excitation of Flavin-Dependent "Ene"-Reductases.

Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor-acceptor complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within "ene"-reductase active sites enables new substrates to participate in photoenzymatic reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism.

Photoenzymatic Hydrogenation of Heteroaromatic Olefins Using 'Ene'-Reductases with Photoredox Catalysts.

Flavin-dependent 'ene'-reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be "dynamically stable", suggesting it is sufficiently long-lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models.

Emerging strategies for expanding the toolbox of enzymes in biocatalysis.

Expanding the repertoire of reactions available to enzymes is an enduring challenge in biocatalysis. Owing to the synthetic versatility of transition metals, metalloenzymes have been favored targets for achieving new catalytic functions. Although less well explored, enzymes lacking metal centers can also be effective catalysts for non-natural reactions, providing access to reaction modalities that compliment those available to metals. By understanding how these activation modes can reveal new functions, strategies can be developed to access novel biocatalytic reactions. This review will cover discoveries in the last two years which access catalytic reactions that go beyond the native repertoire of metal-free biocatalysts.

Photoexcitation of flavoenzymes enables a stereoselective radical cyclization.

Photoexcitation is a common strategy for initiating radical reactions in chemical synthesis. We found that photoexcitation of flavin-dependent "ene"-reductases changes their catalytic function, enabling these enzymes to promote an asymmetric radical cyclization. This reactivity enables the construction of five-, six-, seven-, and eight-membered lactams with stereochemical preference conferred by the enzyme active site. After formation of a prochiral radical, the enzyme guides the delivery of a hydrogen atom from flavin-a challenging feat for small-molecule chemical reagents. The initial electron transfer occurs through direct excitation of an electron donor-acceptor complex that forms between the substrate and the reduced flavin cofactor within the enzyme active site. Photoexcitation of promiscuous flavoenzymes has thus furnished a previously unknown biocatalytic reaction.

Photoenzymatic Catalysis Enables Radical-Mediated Ketone Reduction in Ene-Reductases.

Flavin-dependent ene-reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride-transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non-natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single-electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis.

Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase.

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeBMb) and tuning its heme redox potentials (E°') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme b with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E°' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E°' on NO reduction. Decreasing E°' from +148 to -130 mV significantly impacts electronic properties of the NOR mimics, resulting in 180- and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. Our results indicate that NORs exhibit finely tuned E°' that maximizes their enzymatic efficiency and helps achieve a balance between opposite factors: fast NO binding and decay of dinitrosyl species facilitated by low E°' and fast electron transfer facilitated by high E°'. Only when E°' is optimally tuned in FeBMb(MF-heme) for NO binding, heme-nitrosyl decay, and electron transfer does the protein achieve multiple (>35) turnovers, previously not achieved by synthetic or enzyme-based NOR models. This also explains a long-standing question in bioenergetics of selective cross-reactivity in HCOs. Only HCOs with heme E°' in a similar range as NORs (between -59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E°' in various metalloproteins for their optimal functionality.

Enantioselective Hydrogen Atom Transfer: Discovery of Catalytic Promiscuity in Flavin-Dependent 'Ene'-Reductases.

Flavin has long been known to function as a single electron reductant in biological settings, but this reactivity has rarely been observed with flavoproteins used in organic synthesis. Here we describe the discovery of an enantioselective radical dehalogenation pathway for α-bromoesters using flavin-dependent 'ene'-reductases. Mechanistic experiments support the role of flavin hydroquinone as a single electron reductant, flavin semiquinone as the hydrogen atom source, and the enzyme as the source of chirality.

Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases.

Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

Crystallographic insights into the structure-activity relationships of diazaborine enoyl-ACP reductase inhibitors.

Enoyl-ACP reductase, the last enzyme of the fatty-acid biosynthetic pathway, is the molecular target for several successful antibiotics such as the tuberculosis therapeutic isoniazid. It is currently under investigation as a narrow-spectrum antibiotic target for the treatment of several types of bacterial infections. The diazaborine family is a group of boron heterocycle-based synthetic antibacterial inhibitors known to target enoyl-ACP reductase. Development of this class of molecules has thus far focused solely on the sulfonyl-containing versions. Here, the requirement for the sulfonyl group in the diazaborine scaffold was investigated by examining several recently characterized enoyl-ACP reductase inhibitors that lack the sulfonyl group and exhibit additional variability in substitutions, size and flexibility. Biochemical studies are reported showing the inhibition of Escherichia coli enoyl-ACP reductase by four diazaborines, and the crystal structures of two of the inhibitors bound to E. coli enoyl-ACP reductase solved to 2.07 and 2.11 Šresolution are reported. The results show that the sulfonyl group can be replaced with an amide or thioamide without disruption of the mode of inhibition of the molecule.