Structure of a water soluble fragment of the ‘Rieske’ iron–sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 å resolution

Abstract

Background: The ‘Rieske’ iron–sulfur protein is the primary electron acceptor during hydroquinone oxidation in cytochrome bc complexes. The spectroscopic and electrochemical properties of the ‘Rieske’ [2Fe–2S] cluster differ significantly from those of other iron–sulfur clusters. A 129-residue water soluble fragment containing the intact [2Fe–2S] cluster was isolated following proteolytic digestion of the bc 1 complex and used for structural studies. Results The structure of the Rieske iron–sulfur fragment containing the reduced [2Fe–2S] cluster has been determined using the multiwavelength anomalous diffraction (MAD) technique and refined at 1.5 å resolution. The fragment has a novel overall fold that includes three sheets of β strands. The iron atoms of the [2Fe–2S] cluster are coordinated by two cysteine (Fe-1) and two histidine (Fe-2) residues, respectively, with the histidine ligands completely exposed to the solvent. This is in contrast to the four cysteine coordination pattern observed in previously characterised [2Fe–2S] ferredoxins. The cluster-binding fold is formed by two loops connected by a disulfide bridge; these loops superpose with the metal-binding loops of rubredoxins. The environment of the cluster is stabilised by an extensive hydrogen-bond network. Conclusion The high-resolution structure supports the proposed coordination pattern involving histidine ligands and provides a basis for a detailed analysis of the spectroscopic and electrochemical properties. As the cluster is located at the tip of the protein, it might come into close contact with cytochrome b. The exposed Nϵ atoms of the histidine ligands of the cluster are readily accessible to quinones and inhibitors within the hydroquinone oxidation (Q P) pocket of the bc 1 complex and may undergo redox-dependent protonation/deprotonation.