title: Mutation of the His ligand in mitoNEET stabilizes the 2Fe-2S cluster despite conformational heterogeneity in the ligand environment.
abstract: MitoNEET is the only identified Fe-S protein localized to the outer mitochondrial membrane and a 1.5 Å resolution X-ray analysis has revealed a unique structure [Paddock et al. (2007), Proc. Natl Acad. Sci. USA, 104, 14342-14347]. The 2Fe-2S cluster is bound with a 3Cys-1His coordination which defines a new class of 2Fe-2S proteins. The hallmark feature of this class is the single noncysteine ligand His87, which when replaced by Cys decreases the redox potential (E(m)) by ∼300 mV and increases the stability of the cluster by around sixfold. Unexpectedly, the pH dependence of the lifetime of the 2Fe-2S cluster remains the same as in the wild-type protein. Here, the crystal structure of H87C mitoNEET was determined to 1.7 Å resolution (R factor = 18%) to investigate the structural basis of the changes in the properties of the 2Fe-2S cluster. In comparison to the wild type, structural changes are localized to the immediate vicinity of the cluster-binding region. Despite the increased stability, Cys87 displays two distinct conformations, with distances of 2.3 and 3.2 Å between the S(γ) and the outer Fe of the 2Fe-2S cluster. In addition, Lys55 exhibits multiple conformations in the H87C mutant protein. The structure and distinct characteristics of the H87C mutant provide a framework for further studies investigating the effects of mutation on the properties of the 2Fe-2S cluster in this new class of proteins.
authors.affiliation: Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Conlan
authors.forename: Mark L
authors.initials: ML
authors.lastname: Paddock
authors.forename: Christina
authors.initials: C
authors.lastname: Homer
authors.forename: Herbert L
authors.initials: HL
authors.lastname: Axelrod
authors.forename: Aina E
authors.initials: AE
authors.lastname: Cohen
authors.forename: Edward C
authors.initials: EC
authors.lastname: Abresch
authors.forename: John A
authors.initials: JA
authors.lastname: Zuris
authors.forename: Rachel
authors.initials: R
authors.lastname: Nechushtai
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
mesh_terms: D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D024101:Mitochondrial Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: C525595:CISD1 protein, human; D007506:Iron-Sulfur Proteins; D008024:Ligands; D024101:Mitochondrial Proteins; D006639:Histidine