Generating globin-like reactivities in [human serum albumin-Fe II (heme)] complex through N-donor ligand addition.

Human serum albumin (HSA) has a strong binding affinity for heme b, forming a complex in a 1:1 ratio with the co-factor ([HSA-Fe ^III heme]). This system displays spectroscopic and functional properties comparable to globins when chemical derivatives mimicking them are incorporated into the protein matrix. The aim of this study is to generate globin-like systems using [HSA-Fe ^III heme] as a protein template and binding N-donor ligands (imidazole, Im; and 1-methylimidazole, 1-MeIm) to construct artificial [HSA-Fe(heme)-(N-donor)] complexes. Their electronic structure and binding thermodynamics are investigated using UV-vis and (synchronous) fluorescence spectroscopies, while ligand-protein interactions are visualized using docking simulations. The imidazole derivatives have a strong affinity for [HSA-Fe ^III heme] (K ∼ 10 ⁴ -10 ⁶ ), where the spontaneous binding of Im and 1-MeIm are dominated by entropic and enthalpic effects, respectively. The reduced form of the [HSA-Fe(heme)-(N-donor)] complexes demonstrate nitrite reductase (NiR) activity similar to that observed in globins, but with significant differences in their rates. [HSA-Fe ^II heme-(1-MeIm)] reduces nitrite ∼4× faster than the Im analogue, and ∼ 30× faster than myoglobin (Mb). The enhanced NiR activity of [HSA-Fe ^II heme-(1-MeIm)] is a cumulative effect of several factors including a slightly expanded and more optimal heme binding pocket, nearby residues as possible proton sources, and a H-bonding interaction between 1-MeIm and residues Arg160 and Lys181 that may have a long-distance influence on the heme π electron density.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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