Novel insights into heme binding to hemoglobin.

Abstract

Under hemolytic conditions, hemoglobin and subsequently heme are rapidly released, leading to the toxic effects characterizing diseases such as β-thalassemia and sickle cell disease. Herein, we provide evidence that human hemoglobin can bind heme in a transient fashion via surface-exposed sequence motifs. Following the synthesis of potential heme-binding motifs (HBMs) as peptides, their heme-binding capacity was investigated by UV-vis spectroscopy and ranked according to their binding affinity. Heme binding to human hemoglobin was subsequently studied by UV-vis and surface plasmon resonance (SPR) spectroscopy, revealing a heme-binding affinity in the sub- to micromolar range and a stoichiometry that clearly exceeds a 1:1 ratio. In silico molecular docking and simulation studies confirmed heme binding to the respective motifs in the β-chain of hemoglobin. Finally, the peroxidase-like activity of hemoglobin and the hemoglobin-heme complex was monitored, which indicated a much higher activity (>1800%) than other heme-peptide/protein complexes reported so far. The present study provides novel insights into the nature of intact hemoglobin concerning its transient interaction with heme, which suggests for the first time potential heme-scavenging properties of the protein at concomitant disassembly and, consequently, a potentiation of hemolysis and related processes.