Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions
Yu Feng1, Xi-Chun Liu1, Lianzhi Li2
1School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
Insights
Human cytochrome c (hCyt c) residue Ile81 suppresses peroxidase activity and is vital for binding neuroglobin (Ngb). A mutation at this site enhances peroxidase activity and disrupts hCyt c-Ngb interaction.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Human cytochrome c (hCyt c) is a key heme protein involved in energy conversion and apoptosis.
- Conserved structure of Cyt c has few natural human variants, with position 81 proposed as a key regulatory site.
Purpose of the Study:
- To investigate the role of Ile81 in hCyt c's peroxidase activity and its interaction with neuroglobin (Ngb).
- To elucidate the structure-function relationship of conserved Ile81 in vertebrate Cyt c.
Main Methods:
- Kinetic assays to measure peroxidase activity.
- Analysis of local stability using biophysical methods.
- Alphafold2 prediction for protein-protein interaction modeling.
Main Results:
- Ile81 suppresses hCyt c's intrinsic peroxidase activity.
- The I81N variant exhibits threefold increased peroxidase activity at pH 5.
- Mutation I81N decreases local stability and abolishes the hCyt c-Ngb binding interface, reducing affinity by approximately 40-fold.
Conclusions:
- Ile81 is crucial for modulating hCyt c peroxidase activity and its interaction with Ngb.
- The I81N mutation significantly alters hCyt c's functional and binding properties.
- Findings offer insights into the structure-function dynamics of conserved residues in Cyt c.
Abstract:
Human cytochrome c (hCyt c) is a crucial heme protein and plays an indispensable role in energy conversion and intrinsic apoptosis pathways. The sequence and structure of Cyt c were evolutionarily conserved and only a few naturally occurring mutants were detected in humans. Among those variable sites, position 81 was proposed to act as a peroxidase switch in the initiation stages of apoptosis. In this study, we show that Ile81 not only suppresses the intrinsic peroxidase activity but also is essential for Cyt c to interact with neuroglobin (Ngb), a potential protein partner. The kinetic assays showed that the peroxidase activity of the naturally occurring variant I81N was enhanced up to threefold under pH 5. The local stability of the Ω-loop D (residues 70-85) in the I81N variant was decreased. Moreover, the Alphafold2 program predicted that Ile81 forms stable contact with human Ngb. Meanwhile, the Ile81 to Asn81 missense mutation abolishes the interaction interface, resulting in a ∼40-fold decrease in binding affinity. These observations provide an insight into the structure-function relationship of the conserved Ile81 in vertebrate Cyt c.
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