Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H2O2). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.

The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

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Single‐atom catalysts (SACs) exhibit unparalleled atomic utilization and catalytic efficiency, yet it is challenging to modulate SACs with highly dispersed single‐atoms, mesopores, and well‐regulated coordination environment simultaneously and ultimately maximize their catalytic efficiency. Here, a generalized strategy to construct highly active ferric‐centered SACs (Fe‐SACs) is developed successfully via a biomineralization strategy that enables the homogeneous encapsulation of metalloproteins within metal–organic frameworks (MOFs) followed by pyrolysis. The results demonstrate that the constructed metalloprotein‐MOF‐templated Fe‐SACs achieve up to 23‐fold and 47‐fold higher activity compared to those using metal ions as the single‐atom source and those with large mesopores induced by Zn evaporation, respectively, as well as up to a 25‐fold and 1900‐fold higher catalytic efficiency compared to natural enzymes and natural‐enzyme‐immobilized MOFs. Furthermore, this strategy can be generalized to a variety of metal‐containing metalloproteins and enzymes. The enhanced catalytic activity of Fe‐SACs benefits from the highly dispersed atoms, mesopores, as well as the regulated coordination environment of single‐atom active sites induced by metalloproteins. Furthermore, the developed Fe‐SACs act as an excellent and effective therapeutic platform for suppressing tumor cell growth. This work advances the development of highly efficient SACs using metalloproteins‐MOFs as a template with diverse biotechnological applications.

The mechanism of the Ley–Griffith alcohol oxidation has been elucidated using time-resolved spectroscopic methods.

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Single‐Atom Catalysts In article number 2205674, Kang Liang and co‐workers develop a generalized strategy to construct highly active ferric‐centered single‐atom catalysts via a biomineralization strategy that enables the homogeneous encapsulation of metalloproteins within metal‐organic frameworks followed by pyrolysis. The enhanced activity of the obtained catalysts benefits from the highly dispersed atoms, the mesopores, and the regulated coordination environment of the single‐atom active sites induced by the metalloproteins.

We report the synthesis of a series of ruthenium complexes supported by the phosphine olefin ligand tropPPh2 (trop=5-H-dibenzo-[a,d]cyclohepten-5-yl) in the oxidation states 0, +I, and +II, formed via successive one-electron oxidization steps from Ru(0) (tropPPh2 )2 . The bidentate character of the tropPPh2 ligand and its steric hindrance force the complexes to adopt uncommon geometries, which were investigated by X-ray diffraction analysis. EPR data of the mononuclear Ru(I) complex reveal couplings of the unpaired spin with the ruthenium and two phosphorus nuclei, as well as the olefinic protons which show that the spin is mainly localized on the Ru(I) center.