Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential

Ian J. Campbell, Dimithree Kahanda, Joshua T. Atkinson, Othneil Noble Sparks, Jinyoung Kim, Chia Ping Tseng, Rafael Verduzco, George N. Bennett, Jonathan J. Silberg

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Recombination can be used in the laboratory to overcome component limitations in synthetic biology by creating enzymes that exhibit distinct activities and stabilities from native proteins. To investigate how recombination affects the properties of an oxidoreductase that transfers electrons in cells, we created ferredoxin (Fd) chimeras by recombining distantly related cyanobacterial and cyanomyophage Fds (53% identity) that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required for similar levels of cellular electron transfer. These results show how Fds can be diversified through recombination and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic metabolic pathway.

Original languageEnglish (US)
Pages (from-to)3245-3253
Number of pages9
JournalACS Synthetic Biology
Volume9
Issue number12
DOIs
StatePublished - Dec 18 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Keywords

  • electron transfer
  • ferredoxin
  • midpoint potential
  • recombination
  • sulfite reductase
  • thermostability

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