Unveiling an Ultrareduced State of Heme in Myoglobin.

Heme proteins are essential metalloproteins with diverse biological roles, and these functions are facilitated by the heme's ability to adopt multiple oxidation states, with Fe(II), Fe(III), and Fe(IV) being the most commonly observed. While highly reduced heme states beyond Fe(II) have been studied in synthetic complexes, their presence and characterization in native hemoproteins have remained largely unexplored. In this study, we report a full conversion of sperm whale myoglobin (Mb) to an ultrareduced state through chemical reduction under physiologically relevant conditions. Extensive spectroscopic analyses reveal a concerted proton-coupled two-electron reduction of the porphyrin macrocycle, forming a novel ultrareduced species. The identity of this ultrareduced Mb was determined to be a two-electron reduced product of deoxyMb, Por(2e)Fe(II)-Mb. These experimental data indicate that the reduction reaction is porphyrin ligand-centered, and proton transfer might also be involved, generating a high-spin ( = 2) Fe(II) species in a native heme protein. The electron-rich nature of Por(2e)Fe(II)-Mb enables it to perform a two-electron reduction of NO to NO via a ferrous-nitrosyl intermediate state. These findings expand our understanding of ultrareduced heme iron states in hemoproteins and their potential applications in biochemical and biotechnological fields.