Brustad, E.M.; Nicewicz, D.A.

ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362

A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

Shafaat, H.S.

J. Am. Chem. Soc. 2018, 140, 10250-10262, 10.1021/jacs.8b05194

Well-defined molecular systems for catalytic hydrogen production that are robust, easily generated, and active under mild aqueous conditions remain underdeveloped. Nickel-substituted rubredoxin (NiRd) is one such system, featuring a tetrathiolate coordination environment around the nickel center that is identical to the native [NiFe] hydrogenases and demonstrating hydrogenase-like proton reduction activity. However, until now, the catalytic mechanism has remained elusive. In this work, we have combined quantitative protein film electrochemistry with optical and vibrational spectroscopy, density functional theory calculations, and molecular dynamics simulations to interrogate the mechanism of H2 evolution by NiRd. Proton-coupled electron transfer is found to be essential for catalysis. The coordinating thiolate ligands serve as the sites of protonation, a role that remains debated in the native [NiFe] hydrogenases, with reduction occurring at the nickel center following protonation. The rate-determining step is suggested to be intramolecular proton transfer via thiol inversion to generate a NiIII–hydride species. NiRd catalysis is found to be completely insensitive to the presence of oxygen, another advantage over the native [NiFe] hydrogenase enzymes, with potential implications for membrane-less fuel cells and aerobic hydrogen evolution. Targeted mutations around the metal center are seen to increase the activity and perturb the rate-determining process, highlighting the importance of the outer coordination sphere. Collectively, these results indicate that NiRd evolves H2 through a mechanism similar to that of the [NiFe] hydrogenases, suggesting a role for thiolate protonation in the native enzyme and guiding rational optimization of the NiRd system.

Kim, H.M.; Lee, H.S.

Biomacromolecules 2020, 21, 5021-5028, 10.1021/acs.biomac.0c01194

Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein–protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+ and Cu2+), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.

Shafaat, H.S.

J. Phys. Chem. Lett. 2015, 6, 3731-3736, 10.1021/acs.jpclett.5b01750

A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20–100 s–1 at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.