Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Zhu, Shaotong; Vik, Steven B.

doi:10.1074/jbc.m115.660381

Cited by 28 publications

(35 citation statements)

References 41 publications

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“…Materials were obtained as described in previous work (Zhu and Vik 2015). MTS (Methanethiosulfonate) cross-linking reagents, M3M (1,3-Propanediyl bismethanethiosulfonate), and M6M (1,6-Hexanediyl bismethanethiosulfonate) were from Toronto Research Chemicals (Toronto, Canada).…”

Section: Methodsmentioning

confidence: 99%

“…The polyclonal antibodies against E. coli complex I subunits L and M were prepared by Affinity BioReagents (Golden CO USA). These antibodies were raised in rabbits against peptide QTYSQPLWTWMSVGD (corresponding to residues 58–72) in subunit L and peptide GKAKSQIASQELPGM (corresponding to residues 446–460) in subunit M, and were described previously (Michel et al 2011; Zhu and Vik 2015). Subunit N was detected by the monoclonal rat anti-HA (high affinity) antibody from Roche.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids pUC19-L'MN, 6.57 kb, (Michel et al 2011) and pUC19-L'MN-SpeI, 6.62 kb, (Zhu and Vik 2015) were used for construction of cysteine mutants K581C ( nuoL ) and T292C ( nuoN ), respectively. Both plasmids contain a truncated gene for L and full length genes for subunits M and N. They differ only in that pUC19 L'MN-SpeI contains a unique SpeI endonuclease site downstream of the nuoN gene, and nuoN has HA and His tags that are identical to those in the expression vector pBA400.…”

Section: Methodsmentioning

confidence: 99%

“…Recent work from this lab has shown that constraining the lateral helix of subunit L by a variety of different cross-links did not reduce enzyme activity or proton translocation (Zhu and Vik 2015). In this report we have tried a similar approach to test whether the C-terminus of subunit K has a dynamic role in the enzyme function of Complex I.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…The membrane domain of complex I also contains a long, approximately 110-Å, transverse horizontal helix (HL) from Nqo12 that spans along the domain and interacts with all three antiporter-like subunits. The helix is likely to have a clamping function and forms an element important for structure stability (35)(36)(37).…”

Section: Significancementioning

confidence: 99%

Symmetry-related proton transfer pathways in respiratory complex I

Luca

Gámiz‐Hernández

Kaila

2017

Proc. Natl. Acad. Sci. U.S.A.

170

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Complex I functions as the initial electron acceptor in aerobic respiratory chains of most organisms. This gigantic redox-driven enzyme employs the energy from quinone reduction to pump protons across its complete approximately 200-Å membrane domain, thermodynamically driving synthesis of ATP. Despite recently resolved structures from several species, the molecular mechanism by which complex I catalyzes this long-range protoncoupled electron transfer process, however, still remains unclear. We perform here large-scale classical and quantum molecular simulations to study the function of the proton pump in complex I from Thermus thermophilus. The simulations suggest that proton channels are established at symmetry-related locations in four subunits of the membrane domain. The channels open up by formation of quasi one-dimensional water chains that are sensitive to the protonation states of buried residues at structurally conserved broken helix elements. Our combined data provide mechanistic insight into long-range coupling effects and predictions for sitedirected mutagenesis experiments.NADH:ubiquinone oxidoreductase | proton pumping | Grotthuss mechanism | multiscale simulation | bioenergetics C omplex I (NADH:ubiquinone reductase) is the largest enzyme of the respiratory chain, generating a proton motive force (pmf) that is used for synthesis of adenosine triphosphate (ATP) and active transport (1, 2). Complex I catalyzes electron transfer (eT) between nicotine adenine dinucleotide (NADH) and quinone (Q), and couples the energy released to pumping of four protons across the membrane (3-9). The distance between the electron and proton transferring modules extends up to approximately 200 Å. It currently remains unclear, however, how complex I catalyzes this remarkable long-range proton-coupled electron transfer (PCET) process. In addition to its central role in biological energy conversion, elucidating the molecular mechanism of complex

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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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Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Cited by 28 publications

References 41 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

Symmetry-related proton transfer pathways in respiratory complex I

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

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