Insight into Molecular Stability and Physiological Properties of the Diheme Cytochrome CYC41 from the Acidophilic Bacterium Acidithiobacillus ferrooxidans

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• 作者：Guillaume Malarte ; Gisè ; le Leroy ; Elisabeth Lojou ; Chantal Abergel ; Mireille Bruschi ; and Marie Thé ; rè ; se Giudici-Orticoni
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 3, 2005
• 年：2005
• 卷：44
• 期：17
• 页码：6471 - 6481
• 全文大小：359K
• 年卷期：v.44,no.17(May 3, 2005)
• ISSN：1520-4995

文摘

The cyc1 gene encoding the soluble dihemic cytochrome c CYC₄₁ from Acidithiobacillusferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the hostorganism. The cytochrome was successfully produced and folded only in fermentative conditions: thisallowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at twosequence positions (E121 and Y63) were introduced near the two hemes in order to assign individualredox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin andcytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC₄₁ with biochemical andbiophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environmentof heme B. This result indicates that heme B has the lower redox potential. Interaction studies with thetwo physiological partners indicate that CYC₄₁ functions as an electron wire between RCy and cytochromeoxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interactionwith RCy. A docking analysis of CYC₄₁, RCy, and cytochrome oxidase allowed us to propose a modelfor the complex in agreement with our experimental data.