An artificial metalloenzyme that can oxidize water photocatalytically: design, synthesis, and characterization.

In nature, light-driven water oxidation (WO) catalysis is performed by photosystem II via the delicate interplay of different cofactors positioned in its protein scaffold. Artificial systems for homogeneous photocatalytic WO are based on small molecules that often have limited solubility in aqueous solutions. In this work, we alleviated this issue and present a cobalt-based WO-catalyst containing artificial metalloenzyme (ArM) that is active in light-driven, homogeneous WO catalysis in neutral-pH aqueous solutions. A haem-containing electron transfer protein, cytochrome B5 (CB5), served to host a first-row transition-metal-based WO catalyst, CoSalen (CoIISalen, where H2Salen = N,N'-bis(salicylidene)ethylenediamine), thus producing an ArM capable of driving photocatalytic WO. The CoSalen ArM formed a water-soluble pre-catalyst in the presence of [Ru(bpy)3](ClO4)2 as photosensitizer and Na2S2O8 as the sacrificial electron acceptor, with photocatalytic activity similar to that of free CoSalen. During photocatalysis, the CoSalen-protein interactions were destabilized, and the protein partially unfolded. Rather than forming tens of nanometer sized CoOx nanoparticles as free CoSalen does under photocatalytic WO conditions, the CB5 : CoSalen ArM showed limited protein cross-linking and remained soluble. We conclude that a weak, dynamic interaction between a soluble cobalt species and apoCB5 was formed, which generated a catalytically active adduct during photocatalysis. A detailed analysis was performed on protein stability and decomposition processes during the harsh oxidizing reaction conditions of WO, which will serve for the future design of WO ArMs with improved activity and stability.

A screening method for binding synthetic metallo-complexes to haem proteins.

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV-Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.