Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center

Biochemistry. 2009 Jun 9;48(22):4747-52. doi: 10.1021/bi900028r.


MitoNEET is a 2Fe-2S outer mitochondrial membrane protein that was initially identified as a target for anti-diabetic drugs. It exhibits a novel protein fold, and in contrast to other 2Fe-2S proteins such as Rieske proteins and ferredoxins, the metal clusters in the mitoNEET homodimer are each coordinated by one histidine residue and three cysteine residues. The interaction of the ligating His87 residue with the 2Fe-2S moiety is especially significant because previous studies have shown that replacement with Cys in the H87C mutant stabilizes the cluster against release. Here, we report the resonance Raman spectra of this naturally occurring Fe(2)S(2)(His)(Cys)(3) protein to assess local structural changes associated with cluster lability. Comparison of mitoNEET to its ferredoxin-like H87C mutant indicates that Raman peaks in the approximately 250-300 cm(-1) region of mitoNEET are influenced by the Fe-His87 moiety. Systematic pH-dependent resonance Raman spectral changes were observed in this spectral region for native mitoNEET but not the H87C mutant. The approximately 250-300 cm(-1) region of native mitoNEET is also sensitive to phosphate buffer. Thus, conditions that influence cluster release are shown here to concomitantly affect the resonance Raman spectrum in the region with Fe-His contribution. These results support the hypothesis that the Fe-N(His87) interaction is modulated within the physiological pH range, and this modulation may be critical to the function of mitoNEET.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution / genetics*
  • Cysteine / chemistry*
  • Cysteine / genetics
  • Drug Delivery Systems
  • Histidine / chemistry*
  • Histidine / genetics
  • Humans
  • Hydrogen-Ion Concentration
  • Hypoglycemic Agents / chemistry
  • Hypoglycemic Agents / metabolism
  • Iron-Sulfur Proteins / chemistry*
  • Iron-Sulfur Proteins / genetics
  • Iron-Sulfur Proteins / metabolism
  • Mitochondrial Membranes / chemistry
  • Mitochondrial Membranes / metabolism
  • Mitochondrial Proteins / chemistry*
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Protons
  • Spectrum Analysis, Raman* / methods


  • CISD1 protein, human
  • Hypoglycemic Agents
  • Iron-Sulfur Proteins
  • Mitochondrial Proteins
  • Protons
  • Histidine
  • Cysteine