Development of Mutant C120S Neuroglobin, Its Characteristics and Interaction with Cytochrome c

Objective: The main aim of this work was the development of mutant Ngb C120S, including the production of a 15N-labeled form and its characterization using CD and NMR spectroscopy. Furthermore, we studied the redox reaction between Ngb C120S and Cyt c using stopped-flow spectroscopy. Methods: The C120S mutation was introduced into the NGB gene by site-directed mutagenesis. Recombinant wild-type (WT) and C120S Ngb were expressed in Eschericia coli under both rich and minimal (M9) media conditions, the latter for 15N-labeling. Proteins were purified using a combination of ammonium sulfate precipitation, anion-exchange, and size-exclusion chromatography. Structural and conformational properties were assessed by UV-visible absorption spectroscopy, circular dichroism (CD) spectroscopy, and heteronuclear NMR spectroscopy (1H and 1H–15N HSQC). The kinetics of electron transfer between ferrous Ngb (WT or C120S) and ferric Cyt c were investigated by stopped-flow spectroscopy. Results and Discussion: The C120S mutant was successfully produced with yields and purity comparable to WT Ngb. UV-visible and CD spectra revealed only minor differences between the mutant and WT, indicating that the mutation does not disrupt the overall fold or the heme environment. NMR analysis confirmed that the overall structure and heme hexacoordination were preserved in the mutant. A downfield shift was observed for the 3-CH3 signal for the mutant, as well as significant perturbations in the chemical shifts of approximately 20 cross-peaks, which may be due to minor local rearrangements of hydrogen bonds and/or the chemical environment of neighboring residues at position 120. Stopped-flow kinetic measurements demonstrated that the C120S mutation did not significantly impair electron transfer. Conclusions: The introduction of the C120S mutation has a minor local effect on the physicochemical and structural characteristics of neuroglobin, as well as its interaction with the protein partner, cytochrome c. The mutant neuroglobin will be used to study the structural and dynamic characteristics of the proposed neuroglobin-cytochrome c complex using NMR spectroscopy.