Towards a photo-driven artificial hydrogenase using the biotin-streptavidin technology

It is estimated that the world’s population will reach 11 billion by 2100 and thus the energy need will increase. Already today this is a delicate topic since the use of finite energy sources as coal and oil are depleting. Furthermore, the CO2 released is negatively effecting the climate. Artificial photosynthesis to produce hydrogen as a clean fuel is one possibility that caught much attention. The reaction product of hydrogen and oxygen is solely water. Many small molecule catalysts have already been reported that produce hydrogen, but they are not as active as natural hydrogenases. Natural enzymes have a highly evolved and sophisticated coordination sphere around the catalytic center, as well as hydrogen, proton and electron channels. These features are hardly added by synthetic modification of a ligand of a small molecule catalyst. Incorporation of those catalysts into a protein scaffold would add a second coordination sphere that mimics natural enzymes. In this context, this thesis explores the biocompatibility of some molecular catalysts that produce hydrogen from formic acid for future in vivo protein incorporation applications. Here, we could show that certain complexes show good turnovers and high recyclability rates, as well as oxygen tolerance under bio-compatible reaction conditions. To address the matter of electron transfer in proteins and their surface we explored the electron transfer properties of a dyad of an electron donating triarylamine and a bio-conjugated ruthenium photosensitizer. Different distances of the dyad were tested and the best system was further improved. Not only was an electron acceptor also bio conjugated to the proteins N-terminus, but also a negative patch of three negatively charged amino acids was introduced in close proximity to the photosensitizer. This negative patch increased the local concentration of the electron acceptor, even when not tethered to the protein. We could show that the protein can successively be bio-conjugated in three different ways: i) biotin-binding, ii) nucleophilic substitution on a cysteine residue and iii) N-terminal modification. The compatibility of these biorthogonal bio-conjugation procedures may open a new possibility of assembling catalytic systems on a protein surface. The thesis further discusses the use of the biotin-streptavidin technology to assemble an artificial hydrogenase to perform hydrogen evolution. A small molecule pentapyridin ligated Co catalyst was incorporated into different mutants of streptavidin and photocatalytic hydrogen evolution was measured. It could be shown that a lysine that was incorporated via mutagenesis has a beneficial influence on turnover numbers and rates. We also found that it decreased the initial lagphase, often seen in small molecule hydrogen evolution catalysts, significantly. These findings support the idea that basic residues in close proximity to a hydrogen reducing or oxidizing catalyst have a positive impact, giving insights into its mechanism. The fact that the biotin-streptavidin provides a catalyst incorporated into a protein binding pocket also enables to exclude a heterolytic hydrogen evolution mechanism often proposed, since the Co-centers are too far away to react with each other, at least in our system. We envision that these findings will help to develop artificial hydrogenases that are at least as active as natural hydrogenases