title: Insight into molecular stability and physiological properties of the diheme cytochrome CYC41 from the acidophilic bacterium Acidithiobacillus ferrooxidans.
abstract: The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.
authors.affiliation: Bioénergétique et Ingénierie des Protéines, CNRS, IBSM, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Malarte
authors.forename: Gisèle
authors.initials: G
authors.lastname: Leroy
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lojou
authors.forename: Chantal
authors.initials: C
authors.lastname: Abergel
authors.forename: Mireille
authors.initials: M
authors.lastname: Bruschi
authors.forename: Marie Thérèse
authors.initials: MT
authors.lastname: Giudici-Orticoni
mesh_terms: D042763:Acidithiobacillus / Q000201:enzymology* / Q000235:genetics; D001400:Azurin / Q000737:chemistry; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000235:genetics
chemical_list: D003574:Cytochrome c Group; D011994:Recombinant Proteins; C012543:rusticyanin; D001400:Azurin; C027728:heme a; C034186:cytochrome C4; D014443:Tyrosine; D006418:Heme; D010088:Oxidoreductases; C090099:diheme cytochrome c; D003576:Electron Transport Complex IV