Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis

Andrei Gilep, Alexey Kuzikov, Tatsiana Sushko, Irina Grabovec, Rami Masamrekh, Larisa V. Sigolaeva, Dmitry V. Pergushov, Felix H. Schacher, Natallia Strushkevich, Victoria V. Shumyantseva

Research output: Contribution to journalArticlepeer-review

Abstract

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E0Ꞌ) of mutants did not significantly differ from the wild type protein and vary in the range of −264 to −231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.

Original languageEnglish
Article number140734
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1870
Issue number1
DOIs
Publication statusPublished - Jan 2022

Keywords

  • Amphiphilic diblock copolymer
  • Carbon nanotubes
  • Cytochrome P450
  • Electroanalysis
  • electron transfer
  • Mycobacterium tuberculosis
  • Rubredoxins
  • Screen-printed electrodes

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