PNNL

Abstract

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(P^(Et_2 )NglycineP^(Et_2 ))2] , immobilized covalently within a protein scaffold was activated for CO2 hydrogenation. Here, using an iterative design where the effect of arginine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO2 hydrogenation with seven unique constructs. Single, double, and triple point mutations were introduced to directly compare catalytic activity, as monitored by turn over frequencies (TOFs) measured with operando 1H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild-type, with overall rates ranging from 0.2 to 0.7 hr 1. Molecular dynamics studies suggest that in these two constructs, the positive residues are oriented next to the active site, consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The positioning of the positive charge in the other scaffolds was not oriented next to the metal. Analysis of molecular dynamics (MD) data shows that the positive charge near the active site holds CO2 near the active site. MD of water distributed around the active site suggest that controlling water close to the active site may also contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.