Download MendeleyEffect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c.
Nold, S.M., Lei, H., Mou, T.C., Bowler, B.E.(2017) Biochemistry 56: 3358-3368
- PubMed: 28598148
- DOI: https://doi.org/10.1021/acs.biochem.7b00342
- Primary Citation of Related Structures:
5TY3 - PubMed Abstract:
We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. U. S. A. 111, 6648-6653]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pK a of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 unit. Consistent with the increase in the apparent pK a , the rate of formation of the dominant alkaline conformer decreases, and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases k cat by 1.6-4-fold at pH 7-10, an effect larger than that seen for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the stronger effect of the K72A mutation on the peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human vs Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.
Organizational Affiliation:
Department of Chemistry and Biochemistry, University of Montana , Missoula, Montana 59812, United States.
