Banse, F.; Mahy, J.-P.

Chem. - Eur. J. 2015, 21, 12188-12193, 10.1002/chem.201501755

An artificial metalloenzyme based on the covalent grafting of a nonheme FeII polyazadentate complex into bovine β‐lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the FeII catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin‐state conversion of the initial high spin (S=2) FeII complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center’s first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2O2 reveals the generation of a high spin (S=5/2) FeIII(η2‐O2) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate.

Cuatrecasas, P.

J. Biol. Chem. 1967, n/a

A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mp which accompanies deoxyribonucleic acid and RNA hy- drolysis. Initial velocities are proportional to enzyme con- centration over a 70-fold range. The enzyme has greater aflinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is be- tween 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca+f is required. The inhibitory effect of high Ca+f concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr+f, while Fe++ and C&f cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca+f or Sr+f suggest that these substrates are hy- drolyzed by the same or closely related regions on the en- zyme. Enzyme activity toward DNA and RNA is strongly in- hibited by 5’-phosphoryl (not by 2’- or 3’-phosphoryl) deriva- tives of deoxyadenylic, adenylic, and deozythymidylic acids, and deozythymidine 3’,5’-diphosphate is the most po- tent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These tidings are consistent with the known action of the enzyme (cleavage of the 5’-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the afhnity toward the constituent bases of these nucleic acids.

Arnold, F.H.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 137-170, 10.1002/9783527804085.ch5

Directed evolution is a powerful algorithm for engineering proteins to have novel and useful properties. However, we do not yet fully understand the characteristics of an evolvable system. In this chapter, we present examples where directed evolution has been used to enhance the performance of metalloenzymes, focusing first on “classical” cases such as improving enzyme stability or expanding the scope of natural reactivity. We then discuss how directed evolution has been extended to artificial systems, in which a metalloprotein catalyzes reactions using abiological reagents or in which the protein utilizes a nonnatural cofactor for catalysis. These examples demonstrate that directed evolution can also be applied to artificial systems to improve catalytic properties, such as activity and enantioselectivity, and to favor a different product than that favored by small‐molecule catalysts. Future work will help define the extent to which artificial metalloenzymes can be altered and optimized by directed evolution and the best approaches for doing so.

Hu, X.; Shima, S.

Angew. Chem. Int. Ed. 2021, 60, 13350-13357, 10.1002/anie.202100443

The reconstitution of [Mn]-hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro-base that may participate in metal–ligand cooperative catalysis or have no internal base or pro-base. Only MnI complexes with an internal base or pro-base are active for H2 activation; only [Mn]-hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal–ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]-hydrogenases. Owing to the nature and position of the internal base or pro-base, the mode of metal–ligand cooperation in two active [Mn]-hydrogenases is different from that of the native [Fe]-hydrogenase. One [Mn]-hydrogenase has the highest specific activity of semi-synthetic [Mn]- and [Fe]-hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site.

Arnold, F.H.; Fasan, R.

Curr. Opin. Biotechnol. 2017, 47, 102-111, 10.1016/j.copbio.2017.06.005

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle–Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities.

Lombardi, A.; Louro, R.O.

Chem. Commun. 2021, 57, 990-993, 10.1039/d0cc06676a

Fe-Mimochrome VI*a is a synthetic peroxidase and peroxygenase, featuring two different peptides that are covalently-linked to deuteroheme. To perform a systematic structure/function correlation, we purposely shortened the distance between the distal peptide and the heme, allowing for the separation and characterization of two regioisomers. They differ in both His axial-ligand orientation, as determined by paramagnetic NMR shifts, and activity. These findings highlight that synthetic metalloenzymes may provide an efficient tool for disentangling the role of axial ligand orientation over peroxidase activity.

Arnold, F.H.

J. Am. Chem. Soc. 2019, 141, 19585-19588, 10.1021/jacs.9b11608

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that Pseudomonas savastanoi ethylene-forming enzyme, a non-heme iron enzyme, can catalyze olefin aziridination and nitrene C−H insertion, and that these activities can be improved by directed evolution. The nonheme iron center allows for facile modification of the primary coordination sphere by addition of metalcoordinating molecules, enabling control over enzyme activity and selectivity using small molecules.

Hu, X.; Shima, S.

Nat. Chem. 2015, 7, 995-1002, 10.1038/Nchem.2382

[Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe2+ is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.

Arnold, F.H.

Curr. Opin. Chem. Biol. 2019, 49, 67-75, 10.1016/j.cbpa.2018.10.004

C–H functionalization is an attractive strategy to construct and diversify molecules. Heme proteins, predominantly cytochromes P450, are responsible for an array of C–H oxidations in biology. Recent work has coupled concepts from synthetic chemistry, computation, and natural product biosynthesis to engineer heme protein systems to deliver products with tailored oxidation patterns. Heme protein catalysis has been shown to go well beyond these native reactions and now accesses new-to-nature C–H transformations, including C–N and C–C bond forming processes. Emerging work with these systems moves us along the ambitious path of building complexity from the ubiquitous C–H bond.