Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex <scp>I</scp>

Steimle, Stefan; Schnick, Christian; Burger, Eva-Maria; Nuber, Franziska; Krämer, Dorothée; Dawitz, Hannah; Brander, Sofia; Matlosz, Bartlomiej; Schäfer, Jacob; Maurer, Katharina; Glessner, Udo; Friedrich, Thorsten

doi:10.1111/mmi.13112

Cited by 18 publications

(11 citation statements)

References 31 publications

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“…We have recently provided evidence (Zhu and Vik 2015), through a cross-linking approach, that the lateral helix in the E. coli Complex I does not play a dynamic role in function. The results of others (Belevich et al 2011; Steimle et al 2015) also support that conclusion. Similarly, the results of the cross-linking approach presented in this report, suggest that the C-terminal extension of subunit K does not play a dynamic role in energy transduction or proton translocation.…”

Section: Discussionsupporting

confidence: 69%

“…Subunit L differs from the related subunits M and N in that it has 2 additional TM (transmembrane) helices, which are connected by a long lateral helix that contacts subunits M, N and K (Efremov and Sazanov 2011). The role of the lateral helix has been tested by mutagenesis in several recent studies (Belevich et al 2011; Steimle et al 2011; Steimle et al 2015; Steimle et al 2012; Torres-Bacete et al 2011). Proton translocation likely occurs through four pathways.…”

Section: Introductionmentioning

confidence: 99%

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Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L

Zhu

Canales

Bedair

et al. 2016

J Bioenerg Biomembr

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Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.

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Section: Discussionsupporting

confidence: 69%

Section: Introductionmentioning

confidence: 99%

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L

Zhu

Canales

Bedair

et al. 2016

J Bioenerg Biomembr

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“…When subtracting the spectra before and after addition of NADH, differences of 15–18 cm −1 for the signals of the label visualize experimentally a reorganization of distinct positions within the helix toward a more hydrophobic environment. Previous EPR and fluorescence spectroscopy studies by Steimle et al pointed to a nearly imperceptible movement of the helix upon reduction by NADH.…”

Section: Discussionmentioning

confidence: 97%

“…The movement of the helix was probed with fluorescence and nitroxide labels. Slight conformational changes of the helix during the reduction of the complex by NADH became evident but not a large rearrangement [20]. However, it is possible that the size of the fluorophore and the nitroxide label influenced the conformational flexibility of the helix.…”

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confidence: 99%

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS

et al. 2019

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Conformational movements play an important role in enzyme catalysis. Respiratory complex I, an L‐shaped enzyme, connects electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation through its membrane arm by a coupling mechanism still under debate. The amphipathic helix across the membrane arm represents a unique structural feature. Here, we demonstrate a new way to study conformational changes by introducing a small and highly flexible nitrile infrared (IR) label to this helix to visualize movement with surface‐enhanced IR absorption spectroscopy. We find that labeled residues K551CL and Y590CL move to a more hydrophobic environment upon NADH reduction of the enzyme, likely as a response to the reorganization of the antiporter‐like subunits in the membrane arm.

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“…1). Cross-linking studies [6], and the results of several other approaches [7–11], have indicated that this helical element is likely to have a structural role, rather than a dynamic role in proton translocation. Apart from this extra structural component of subunit L, each of these three subunits consists of 14 α-helical TM spans.…”

Section: Introductionmentioning

confidence: 99%

Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking

Tursun

Zhu

Vik

2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.

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Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I

Cited by 18 publications

References 31 publications

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS

Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking

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