The X‐ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand‐binding sites

Abstract

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46 C55) and the engineered (C15 C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Exploiting and Engineering Neuroglobin for Catalyzing Carbene N–H Insertions and the Formation of Quinoxalinones

“…Human neuroglobin (Ngb) contains a heme group with the coordination of His64 and His96, where Cys46 and Cys55 form a disulfide bond, with a free Cys120 . By introducing an additional Cys15, the A15C Ngb mutant forms both the native (C46–C55) and de novo (C15–C120) disulfide bonds, as confirmed by the X-ray crystal structure (Figure A) . A15C Ngb was found to be hyperthermostable ( T m > 100 °C), which offers an ideal protein scaffold for enzyme design.…”

“…22 By introducing an additional Cys15, the A15C Ngb mutant forms both the native (C46−C55) and de novo (C15−C120) disulfide bonds, as confirmed by the X-ray crystal structure (Figure 1A). 23 A15C Ngb was found to be hyperthermostable (T m > 100 °C), 24 which offers an ideal protein scaffold for enzyme design. Recently, multifunctional peroxidase was engineered based on A15C Ngb by modification of the heme active site, which is applicable for dehalogenation and dye decolorization.…”

“…(A) X-ray crystal structure of A15C Ngb (PDB 7VQG), showing the heme coordination state, the native and de novo disulfide bonds, and the binding of 1,4-dioxane to the heme distal pocket. (B) Yields of the N–H insertion product of aniline with EDA catalyzed by WT Ngb and its mutants.…”

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

“…[11] The third cysteine (Cys120) is remote from the heme center. By introducing a cysteine close to Cys120 at position 15 (A15C mutation), a disulfide bond of Cys15-Cys120 could be formed, [12] which enhanced the protein thermal stability. [13] By alteration of the heme ligand such as replacing His64 with a non-coordinating amino acid (Ala/Ser/Glu, etc.…”

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.…”