Designing Metalloenzymes by Controlling the First and Second Coordination Spheres Using Non-Traditional Design Strategies

Abstract

This thesis extends or introduces innovative approaches to de novo design to interrogate metal binding, structure, and catalytic activity using a copper nitrite reductase (CuNiR) model. These designs will be used to investigate, at a molecular level, how the first and second coordination spheres influence the copper’s coordination number and, in turn, modify nitrite reduction in a well-folded 3-stranded α-helical coiled coil (3SCC). The non-coded amino acids 3’Pyridyl alanine [3’Py] and 4’Pyridyl alanine [4’Py] side chains were first designed into the 3SCC forming sequence [Ac-G WKALEEK (LKALEEK)2 PyKALEEK G-NH2]3 (i.e., TRIW) to enforce the-nitrogen of histidine within the core of the assembly (i.e., authentic pyridine type nitrogens). The pyridyl sites formed 3-coordinate cuprous sites and 4- or 5-coordinate cupric geometries which yielded an unnatural type 2 copper center (T2Cu). The cuprous state favored a more 2-coordinate linear geometry by designing space above the active site through the mutation of the leucine layer to an alanine layer (L19A), highlighting the impact of distant residues on protein structure around a metal. CuNiR kinetics were investigated under conditions favoring the binding of nitrite to the cuprous state (i.e., Abraham’s mechanism) which is the alternate, unexplored pathway of native CuNiRs. Compared to the natural histidine site, the pyridine ligands enhanced CuNiR activity by a factor of 400. Interestingly, while the kcat values were maximized (~3 s-1), the catalytic efficiency was doubled (kcat/Km= 5 M-1 s-1 vs. 11 M-1 s-1) which depended on the ligand’s orientation within the active site. The catalytic efficiency is further enhanced by shifting to a 2-coordinate Cu(I) enzyme which resulted in a decrease in the Km from 600 mM to 50 mM and kcat/Km of 39 M-1 s-1. Detailed structural experiments and inhibition kinetics resulted in competitive inhibition between Cl and NO2 providing strong evidence of nitrite binding to the reduced enzyme during the catalytic cycle. This demonstrated the formation of a Cu(I)NO2 adduct and highlighted the Cu(I) enzyme as the important oxidation state controlling catalytic efficiency. It is argued that NO2 displaces one pyridine ligand in the 3-coordinate cuprous complex (verses forming a 4-coordinate Cu(I)(Py)3-NO2), analogous to what is observed with the Cu(I)Cl adducts as a result of the steric constraints of the interior of the core. Collectively, the rationale for a 3-coordinate Cu(I)-NO2 enzyme-substrate complex is explored within the context of the sterics between the primary and secondary coordination spheres. This work will also describe an alternative, novel strategy to repack side chains within the 3SCC using 3-residue non-canonical inserts (i.e., stammer) to overwind the coiled coil to explore nitrite reduction. Stammers will be explored in terms of their ability to change the metal-coordination number or rotate solvent-exposed residues (i.e., glutamate) into the core in proximity of the copper center. While this approach generates 3-coordinate cuprous enzymes for both the 3’Py and 4’Py ligands, 2-coordinate resting states are achieved by implementing two different stammers for either ligand (i.e., AEA and AE3’Py), all of which are distinct from the cuprous states of the Leu/Ala designs in terms of their electronic structure. Each stammer explored resulted in slight variations in the kinetics which impacted only the Km values in the range of 100-400 mM. This study will emphasize yet another approach to controlling the coordination number of copper active sites to tune the catalytic efficiency.