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Serine Mutation on Amicyanin Reveals Functional and Structural Roles of Asn47 and Asn54 in the Cu-Binding Ligand

Eunjeong Kim1, Hyojin Jeong2, Heejin Nam1

  • 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju 61186, Republic of Korea.

Journal of Microbiology and Biotechnology
|December 17, 2025
PubMed

Insights

Mutations in amicyanin, a copper protein, reveal key roles for specific amino acids. Asn47 enhances binding affinity to methylamine dehydrogenase, while Asn54 regulates electron transfer by modifying redox potential.

Area of Science:

  • Biochemistry
  • Protein Science
  • Electron Transfer

Background:

  • Amicyanin is a type 1 copper protein crucial for electron transfer in *Paracoccus denitrificans*.
  • It facilitates electron transfer between methylamine dehydrogenase (MADH) and cytochrome c-551i.
  • Understanding amicyanin's structure-function relationship is vital for elucidating biological electron transport mechanisms.

Purpose of the Study:

  • To investigate the functional roles of Asn47 and Asn54 residues within the copper-binding ligand loop of amicyanin.
  • To determine how mutations at these positions affect amicyanin's spectral and redox properties, protein stability, and interaction kinetics with MADH.

Main Methods:

  • Site-directed mutagenesis to introduce Ser substitutions at Asn47 (N47S) and Asn54 (N54S).
  • Spectroscopic and redox potential measurements to characterize mutant proteins.
  • Assessment of protein stability under thermal and oxidative stress.
  • Kinetic analysis of electron transfer reactions with methylamine dehydrogenase.
  • Computational simulations to model the structural and electronic effects of mutations.

Main Results:

  • N47S amicyanin showed similar redox potential and stability to native amicyanin but significantly improved binding affinity to MADH.
  • N54S amicyanin exhibited a more positive redox potential, reduced stability, and altered kinetics with increased electron transfer but decreased binding affinity.
  • Computational analysis indicated that Asn54 substitution affects the copper active site geometry via H-bond pattern changes, while Asn47 mutation influences amicyanin-MADH interaction by altering loop conformation.

Conclusions:

  • Asn47 is critical for amicyanin's binding affinity to methylamine dehydrogenase.
  • Asn54 plays a regulatory role in electron transfer by modulating the active site's redox midpoint potential.
  • These findings highlight the distinct functional roles of specific residues in amicyanin for efficient electron transfer processes.
Keywords:
AmicyaninCu centercupredoxinelectron transferprotein engineeringredox potential

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