Design and engineering of an artificial disulfide bond in human cytochrome c to regulate the protein structure and function†

Abstract

Human cytochrome c (hCyt c) contains a covalently attached heme group with six-coordination (Met/His) and plays vital biological functions, including electron transfer and peroxidase activity by structural alterations, as well as other functions by interactions with partners such as neuroglobin (Ngb). In this study, we designed and engineered an artificial disulfide bond in hCyt c via double mutations (A51C/G77C) which bridges the Ω-loops C and D. The formation of the intramolecular disulfide bond (Cys51–Cys77) was confirmed by mass spectrometry. The molecular modeling study showed that the disulfide bond did not alter the overall structure, and the local structure where Cys51 was located was well folded into an α-helix. Spectroscopic studies were also performed to probe the effects of the disulfide bond on the protein structure, which revealed that the heme coordination of Met80 was likely weakened. Consequently, the rate of ligand binding and the peroxidase activity were enhanced. Meanwhile, the interaction between hCyt c and Ngb was weakened, as suggested by titration studies. These observations indicate that the dynamic properties of Ω-loops C and D may favor the heme coordination and protein–protein interactions by conformational change, which supports the native functions of hCyt c.