Operando spectroscopy investigations of the redox reactions in heme and heme-proteins

Abstract

Operando spectroscopic investigations during molecular redox processes provide unique insights into complex molecular structures and their transformations. Herein, a combination of a potentiodynamic method with spectroscopy has been employed to holistically investigate the structural transformations during Fe-redox (Fe³⁺ ↔ Fe²⁺) of hemin vis á vis heme-proteins, e.g. myoglobin (Mb), hemoglobin (Hb) and cytochrome-C (Cyt-C). The UV-vis findings reveal the formation of hemozoin (≈heme-dimer), which can be selectively prevented via a high concentration of strongly interacting ligands, e.g. histidine (the fifth coordinating ligand in the heme-based protein). On the other hand, methionine does not prevent the formation of hemozoin. In Mb, Hb, and Cyt-C, as the fifth coordination site is occupied by histidine, hemozoin formation is inhibited. During Fe³⁺→ Fe²⁺, operando circular dichroism exhibits a decrease in the initial helical component in Hb from nearly 40% to 28%, which is close to the initial helix component of Mb (≈25%), strongly indicating denaturation of the protein in the redox pathway. The rate of change of the helices versus potential is almost identical for Mb and Hb, but comparatively faster than Cyt-C. In addition, from the Raman bands of M–N dynamics and protein agglomeration, it is concluded that Cyt-C prefers to agglomerate in the 2+ state, whereas Mb/Hb in the 3+ state. In this report, the power of operando spectroscopy is utilized to unearth the dynamics of hemin and heme-based proteins for comprehending the underlying complexities associated with the molecular redox, which have deep implications in electrocatalysis, energy storage, and sensing.