The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.

Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH : ubiquinone oxidoreductase (complex I) / S. Burschel, D. Kreuzer Decovic, F. Nuber, M. Stiller, M. Hofmann, A. Zupok, B. Siemiatkowska, M. Gorka, S. Leimkuhler, T. Friedrich. - In: MOLECULAR MICROBIOLOGY. - ISSN 0950-382X. - 111:1(2019 Jan), pp. 31-45. [10.1111/mmi.14137]

Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH : ubiquinone oxidoreductase (complex I)

B. Siemiatkowska;
2019

Abstract

The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.
Carrier Proteins; Centrifugation, Density Gradient; Electron Spin Resonance Spectroscopy; Electron Transport Complex I; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Iron-Sulfur Proteins; Protein Multimerization
Settore BIO/11 - Biologia Molecolare
gen-2019
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/704876
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