Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor

Wellinson G. Guimarães, Ana C.S. Gondim, Pedro Mikael da Silva Costa, Marie Alda Gilles-Gonzalez, Luiz G.F. Lopes, Marta S.P. Carepo, Eduardo H.S. Sousa

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN) form was monitored by UV–visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV–visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8 kJ mol− 1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2 kJ mol− 1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.

Original languageEnglish (US)
Pages (from-to)129-137
Number of pages9
JournalJournal of Inorganic Biochemistry
Volume172
DOIs
StatePublished - Jul 1 2017

Keywords

  • Conformational changes
  • FixL
  • Heme-based sensor
  • Oxygen sensor
  • Stability studies
  • Urea

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

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