Apfel, U.-P.; Happe, T.; Kurisu, G.

Chem. Sci. 2016, 7, 959-968, 10.1039/C5SC03397G

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.

Lubitz, W.; Reijerse, E.

Biochemistry 2015, 54, 1474-1483, 10.1021/bi501391d

[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S]2–) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66–70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607–610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN– ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN– ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme.

Onoda, A.

ACS Catal. 2014, 4, 2645-2648, 10.1021/cs500392e

The hydrogen-evolving diiron complex, (μ-S)2Fe2(CO)6 with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)2Fe2(CO)6 core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H2 via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C.

Happe, T.

Nat. Chem. Biol. 2013, 9, 607-609, 10.1038/Nchembio.1311

Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H2-producing catalysts.