Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

Despite CYP102A1 (P450BM3) representing one of the most extensively researchedmetalloenzymes,crystallisation of its haem domain upon modification can be ac hallenge. Crystal structures are indispensable for the efficient structurebased design of P450BM3 as ab iocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2hours and diffracting to an ear-atomic resolution of 1.22 . Using these crystals as seeds in across-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoym olecules and diverse artificial metalloporphyrins binding various ligand molecules,a sw ell as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.

Die Kristallisation der Hämdomäne von CYP102A1 (P450BM3) kann nach Modifizierung eine Herausforderung sein. Dennoch sind solche Kristallstrukturen unentbehrlich für die wirksame strukturbezogene Entwicklung von P450BM3 als Biokatalysator. Es wurde gezeigt, dass das Abietanditerpenderivat N‐Abietoyl‐l‐tryptophan (AbiATrp) ein hervorragender Kristallisationsbeschleuniger ist. Die Nutzung von Kristallen der P450BM3‐Hämdomäne mit AbiATrp als Impfkristalle ermöglichte die rasche Mikrokristallisation bei der naszierende Kristalle innerhalb von Sekunden nach Impfung erschienen. Hierdurch wurde eine Auswahl an Kristallen der P450BM3‐Hämdomäne erhalten, die bisher nicht kristallisierbare Täuschmoleküle, diverse künstliche Metalloporphyrine, welche verschiedenartige Liganden binden, sowie zusätzlich stark getaggte Hämdomänevarianten enthalten (37 zusätzliche Aminosäuren). Manche der Strukturen könnten als Modellverbindungen verschiedener Stadien des katalytischen Zyklus von P450BM3 genutzt werden.

The self-sufficient cytochrome P450 102A1 (P450BM3), known for its highly efficient activation of molecular oxygen, was reconstituted with manganese protoporphyrin IX. This artificial enzyme (Mn-BM3) was found to catalyze the monooxygenation of various substrates by activating molecular oxygen. Investigation using mechanistic probes (i.e., radical clock substrates) revealed that hydroxylation by Mn-BM3 proceeds via a radical intermediate of the substrate, supporting the involvement of an "H atom abstraction/ • OH recombination" mechanism, which is the commonly accepted mechanism for hydroxylation by P450s. A DFT study also indicated that the activation barrier of Mnporphine in the rebound step is higher than that of Fe-porphine, consistent with the experimental results obtained in the radical clock experiment, wherein Mn-BM3 presents a slower rebound rate than that of heme-bound BM3.

Silicon naphthalocyanine nanoparticles were successfully prepared by laser ablation in liquid. Silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) powders in deionized water were irradiated with nanosecond-pulsed laser (Nd:YAG, SHG) to prepare nanoparticles. The prepared nanoparticles were investigated by scanning electron microscopy, dynamic light scattering and spectrophotometry. The shape was polygonal and partially spherical. The primary and secondary particle sizes were reduced with an increase in laser fluence. Absorbance was increased at low laser fluence and was constant at high laser fluence with the increase in laser fluence. The ratio of the two absorbance peaks was changed with the increase in laser fluence.

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.

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Enzymes. In their Research Article (e202215706), Hiroshi Sugimoto, Osami Shoji et al. report the crystal structure of cytochrome P450BM3 mimicking the state of the enzyme in the moment preceding epoxidation.