Borovik, A.S.; Hendrich, M.P.; Moënne-Loccoz, P.

J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Albrecht, M.; Paradisi, F.

Angew. Chem. Int. Ed. 2018, 130, 10837-10842, 10.1002/ange.201807168

Im Tausch gegen NHC: Die Einfügung eines N‐heterocyclischen Carbenliganden (grün/blau) als Ersatz für His in das aktive Zentrum des Redoxenzyms Azurin rekonstituiert das T1‐Kupferzentrum. Der resultierende Komplex ist spektroskopisch kaum unterscheidbar von der N‐Bindung von His oder N‐Methylimidazol, senkt aber signifikant das Reduktionspotential des Kupferzentrums und erleichtert dadurch Elektronentransferprozesse.

Jaussi, R.

Protein Expression Purif. 2014, 93, 54-62, 10.1016/j.pep.2013.10.015

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650 mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2 molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.