Development of Mutant Forms of Neuroglobin with Substitutions in the Interaction Surface with Cytochrome c

Mutant forms of human neuroglobin that carry targeted mutations in the putative interface with cytochrome c: single (E60K, K67E, E87K, K95E) and double (E60K\E87K) substitutions were obtained by site-directed mutagenesis. The E60K, K95E, and E60K\E87K mutations cause slight changes in the UV-vis absorption spectra, which can be associated with both a change in the electrostatic field near the heme and a change in the heme iron spin to the high-spin state. The secondary structure of mutant neuroglobins calculated from the CD spectral data almost did not differ from the secondary structure of wild-type neuroglobin, except for the protein with the K67E substitution, whose beta-turn is reorganized into an alpha-helix. The IR spectra provide further evidence for the predominance of alpha-helices in protein secondary structure for mutant forms of neuroglobin. Thus, the introduction of these mutations did not have a significant effect on the characteristics of the heme-containing protein neuroglobin. The developed mutant forms will be used to study the contribution of individual amino acid residues to the formation of the reaction complex between neuroglobin and cytochrome c, which will allow rational design of drugs for the therapy of various diseases associated with neuronal death in the future.