Constructing Artificial Globin-like Active Sites Using Human Serum Albumin Heme and Testing for their Nitrite Reductase Activity

Abstract:

Human serum albumin (HSA) is found within blood plasma and is a repository for many molecules, such as heme (Fe protoporphyrin IX). Crystal studies show that HSA reconstituted with heme (HSA-heme) generates a Fe-O(Tyr) (Tyr = tyrosine) weakly bonding interaction. This is different from other heme proteins, such as myoglobin (Mb), which has an Fe-N(His) (His = histidine) bond. Previous work, as well as data from our lab, demonstrates that HSA-heme readily forms complexes with N-donor ligands, [HSA-heme-N(ligand)], presumably forming a Fe-N(donor) interaction like that of globins. The goal of this project is to: 1) generate a set of HSA-heme systems complexed with various N-donor ligands (from imidazole derivatives such as imidazole (Im) and methylimidazole (1-MeIm)), which will serve as artificial enzymes mimicking the active site of Mb, and 2) investigate their spectroscopic and thermodynamic properties, and globin-like chemistries such as nitrite reductase (NiR) activity. The latter involves the reduction of NO^2- to NO via Fe^II heme enzyme. Preliminary temperature-dependence fluorescence work shows significant binding of 1-methylimidazole (1-MeIm) to HSA, with a K of ~ 1 x 10⁶ M^-1. The NiR activity of [Fe^II HSA-heme-1-MeIm] demonstrates a rate constant of 0.2 M^-1 s^-1, which is smaller than wild-type FeII HSA-heme (1.3 M^-1 s^-1) or FeII Mb (2.2 M^-1 s^-1). These results will allow us to correlate the electronic structure of the N-donor ligand (1-MeIm versus Im) on the thermodynamics of binding to HSA-heme. Furthermore, they will allow us to understand how the N-donor ligand fine tunes the NiR reactivity of heme enzymes.