Reconstitution of [Fe]-Hydrogenase with Model Complexes Reveals Functional Roles of Methyl Groups in the Metallocofactor.

While [Fe]-hydrogenase represents a promising biological alternative to noble-metal hydrogenation catalysts, difficulties in its production and genetic manipulation significantly hinder both mechanistic investigations and practical applications. Semisynthetic [Fe]-hydrogenases, assembled from recombinant apoenzymes and synthetic FeGP cofactor mimics, address these limitations, yet early-generation semisynthetic [Fe]-hydrogenases suffer from low activity due to incomplete pyridinol ligand features on the FeGP cofactor mimics. Here, we investigate the roles of 3- and 5-methyl substituents on the FeGP cofactor's pyridinol ligand by designing and characterizing model complexes 2-4. Reconstitution studies demonstrate that the 3-methyl group boosts catalytic activity (a 35-fold increase over nonmethylated analogs) and reconstitution kinetics, while the 5-methyl group enhances oxidative stability. The optimized variant, jHmd-4, achieves specific activities of 38.5 (forward) and 33.5 U·mg-1 (reverse), reaching 8% of native enzyme activity─the highest reported for semisynthetic systems to date. This breakthrough establishes a tunable platform for developing efficient biohydrogenation catalysts, while providing key insights into metalloenzyme engineering.