Histidine ligand variants of a flavo-diiron protein: Effects on structure and activities

Han Fang, Jonathan D. Caranto, Rosalinda Mendoza, Alexander B. Taylor, P. John Hart, Donald M. Kurtz

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O 2R). While most FDPs show similar structural, spectroscopic, and redox properties, O2R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O 2R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and -A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O2R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O2 were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O2R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O2R and NOR mechanisms of FDPs.

Original languageEnglish (US)
Pages (from-to)1231-1239
Number of pages9
JournalJournal of Biological Inorganic Chemistry
Issue number8
StatePublished - Dec 2012


  • Dioxygen
  • Enzyme kinetics
  • Nitric oxide
  • Non-heme iron
  • Site-directed mutagenesis
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry


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