A Proximal Arginine R206 Participates in Switching of the Bradyrhizobium japonicum FixL Oxygen Sensor

Marie-Alda Gilles-Gonzalez, Ana Isabel Caceres, Eduardo Henrique Silva Sousa, Diana R Tomchick, Chad A Brautigam, Constancio Gonzalez, Mischa Machius

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd∼350 μM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the FeIII form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.

Original languageEnglish (US)
Pages (from-to)80-89
Number of pages10
JournalJournal of Molecular Biology
Volume360
Issue number1
DOIs
StatePublished - Jun 30 2006

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Keywords

  • FixJ
  • PAS domain
  • response regulator
  • sensor kinase
  • two-component system

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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