Engineering Human Neuroglobin into a Cytochrome c‐Like Protein with a Single Thioether Bond in Non‐native State

Abstract

A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO 2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.

Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin

“…For example, Cyt c-like CÀ S bonds were engineered into Cyt b 5 / b 562 , [14] Mb, [15] and neuroglobin (Ngb). [16] In a previous study, an artificial MnÀ salen complex was covalently linked to the protein scaffold of Mb, which enhanced the enantioselectivity for sulfur oxidation. [17] In parallel with these progresses, multiple strategies have been developed to regulate the heme active site by tuning non-covalent interactions such as hydrogen (H)-bonding and hydrophobic interactions.…”

“…Moreover, artificial cross‐links can be engineered into proteins to stabilize the heme group or other metal cofactors. For example, Cyt c ‐like C−S bonds were engineered into Cyt b 5 / b 562 , [14] Mb, [15] and neuroglobin (Ngb) [16] . In a previous study, an artificial Mn−salen complex was covalently linked to the protein scaffold of Mb, which enhanced the enantioselectivity for sulfur oxidation [17] …”

Self-oxidation of cysteine to sulfinic acid in an engineered T67C myoglobin: structure and reactivity

“…[5][6][7] In cytochrome P450 (CYP450), Cys acts as a proximal ligand for the heme cofactor to activate O 2 , 8 and in human neuroglobin (Ngb) Cys46 and Cys55 form an intramolecular disulfide bond to regulate the ligand binding. [9][10][11][12] Moreover, the third Cys120 in human Ngb may also regulate the intracellular levels of RO/N/S species. 13 As an O 2 carrier, human myoglobin (Mb) possesses a single Cys110 and efficient reduction of the Cys-thiyl radical was observed by electron transfer from glutathione, whereas other mammalian Mbs rarely have Cys residues.…”