Characterization of the Membrane Domain Subunit NuoJ (ND6) of the NADH-Quinone Oxidoreductase from Escherichia coli by Chromosomal DNA Manipulation

详细信息    查看全文

• 作者：Mou-Chieh Kao ; Salvatore Di Bernardo ; Eiko Nakamaru-Ogiso ; Hideto Miyoshi ; Akemi Matsuno-Yagi ; and Takao Yagi
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 8, 2005
• 年：2005
• 卷：44
• 期：9
• 页码：3562 - 3571
• 全文大小：487K
• 年卷期：v.44,no.9(March 8, 2005)
• ISSN：1520-4995

文摘

The ND6 subunit is one of seven mitochondrial DNA-encoded subunits of the proton-translocating NADH-quinone oxidoreductase (complex I). Physiological importance of the ND6 subunitis becoming increasingly apparent because a number of mutations leading to amino acid changes in thissubunit have been found to be associated with known mitochondrial diseases. Using the Escherichia colienzyme (NDH-1), we have investigated the NuoJ subunit (the E. coli counterpart of ND6) by employinga chromosomal DNA manipulation technique. A series of point mutations was constructed directly on thenuoJ gene in the chromosome targeting at highly conserved residues. Analyses with blue-native gelelectrophoresis and immunological methods revealed that, in all point mutants, the assembly of NDH-1was normal and that the deamino-NADH-K₃Fe(CN)₆ reductase activity of the membrane was essentiallythe same as that of the wild-type. However, energy-coupled NDH-1 activities were affected to variedextents. Among them, mutants of the Val-65 residue that is located in the most conserved transmembranesegment significantly lost the coupled electron-transfer activities and exhibited diminished membranepotential and proton translocation. This may suggest that Val-65 or the area around it is important forenergy transduction of the coupling site 1. Together with the results on mutations related to human diseases,possible functional roles of the NuoJ subunit have been discussed.