Characterization of Bacterial Complex I (NDH-1) by a Genetic Engineering Approach

Yagi, Takao; Torres‐Bacete, Jesús; Sinha, Prem Kumar; Castro-Guerrero, Norma; Matsuno‐Yagi, Akemi

doi:10.1007/978-94-007-4138-6_8

A Structural Perspective on Respiratory Complex I 2012

DOI: 10.1007/978-94-007-4138-6_8

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Characterization of Bacterial Complex I (NDH-1) by a Genetic Engineering Approach

Takao Yagi

Jesús Torres‐Bacete

Prem Kumar Sinha

et al.

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Cited by 2 publications

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“…Moreover, there are certain merits in the bacterial system over mitochondrial complex I including the simpler structure, the absence of “assembly factors” and “accessory subunits”, and no potential implications derived from protein and cofactor import that require ATP and the membrane potential. 36 , 37 …”

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confidence: 99%

“…In a series of work, we have established the advantage of the chromosomal DNA manipulation technique and have shown that E. coli NDH-1 is ideally suited to study both membrane and peripheral domains of complex I. − ,,, Our method has an advantage of avoiding polar effects seen in in trans complementation. Moreover, there are certain merits in the bacterial system over mitochondrial complex I including the simpler structure, the absence of “assembly factors” and “accessory subunits”, and no potential implications derived from protein and cofactor import that require ATP and the membrane potential. , …”

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confidence: 99%

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Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

Sinha

Castro-Guerrero²,

Patki³

et al. 2015

Biochemistry

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The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217–Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed.

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confidence: 99%

mentioning

confidence: 99%

Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

Sinha

Castro-Guerrero²,

Patki³

et al. 2015

Biochemistry

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Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation

Sato

Torres‐Bacete

Sinha

et al. 2014

J Bioenerg Biomembr

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The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is the first and largest enzyme of the respiratory chain which has a central role in cellular energy production and is implicated in many human neurodegenerative diseases and aging. It is believed that the peripheral domain of complex I/NDH-1 transfers the electron from NADH to Quinone (Q) and the redox energy couples the proton translocation in the membrane domain. To investigate the mechanism of the proton translocation, in a series of works we have systematically studied all membrane subunits in the Escherichia coli NDH-1 by site-directed mutagenesis. In this mini-review, we have summarized our strategy and results of the mutagenesis by depicting residues essential for proton translocation, along with those for subunit connection. It is suggested that clues to understanding the driving forces of proton translocation lie in the similarities and differences of the membrane subunits, highlighting the communication of essential charged residues among the subunits. A possible proton translocation mechanism with all membrane subunits operating in unison is described.

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Characterization of Bacterial Complex I (NDH-1) by a Genetic Engineering Approach

Cited by 2 publications

References 100 publications

Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation

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