Cytochromes c555 from the Hyperthermophilic Bacterium Aquifex aeolicus. 2. Heterologous Production of Soluble Cytochrome c555s and Investigation o

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• 作者：Corinne Aubert ; Franç ; oise Guerlesquin ; Pierre Bianco ; Gisè ; le Leroy ; Pascale Tron ; Karl-Otto Stetter ; and Mireille Bruschi
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：November 13, 2001
• 年：2001
• 卷：40
• 期：45
• 页码：13690 - 13698
• 全文大小：139K
• 年卷期：v.40,no.45(November 13, 2001)
• ISSN：1520-4995

文摘

The cycB2 gene encoding the soluble cytochrome c₅₅₅^s from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism. The cytochromewas successfully produced in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genesinvolved in the cytochrome c maturation process. Comparison of native and recombinant cytochromec₅₅₅^s shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties.Since two different methionine residues are present in the sequence stretch usually providing the sixthligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionineserving as the axial ligand. Two single mutations were introduced, leading to the replacement of eachmethionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c₅₅₅^s,using biochemical and biophysical techniques has been carried out. The M84H mutant exhibits spectralfeatures identical to those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. Bycontrast, substitution of methionine 78 by a histidine residue strongly alters the structural andphysicochemical properties of the molecule which exhibits characteristics of His/His iron coordinationtype rather than His/Met. These results allow us to identify methionine 78 as the sixth ligand of cytochromec₅₅₅^s heme iron. Preliminary results on the thermostability of the native and mutant cytochromes c₅₅₅ arealso reported.