Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

Abstract

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of (1)H/(2)H exchange kinetics at the level of the amide proton in the mid-infrared spectral range (1700-1500 cm(-1)). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H-bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300-30 cm(-1)), a spectral range that includes a continuum mode of the hydrogen bonding signature.

Involvement of Acidic Amino Acid Residues in Zn2+ Binding to Respiratory Complex I

“…It is likely that this protein moiety is part of the membrane arm as suggested for the un-exchangeable fraction of complex Ii dentified ChemBioChem 2015, 16,2080 -2085 www.chembiochem.org by H/D exchange kinetics of the amide Is ignal. [29] The same holds for the amide II band (1556(À)( WT), 1559(À)c m À1 (histagged)) and amide II' band (1488(À), 1453(À)c m À1 ). These signals barely shifted after H/D exchange.…”

“…This time is necessary to ensure that the solvent-accessible, exchangeable water molecules bound to complex I [29] are fully exchanged. This time is necessary to ensure that the solvent-accessible, exchangeable water molecules bound to complex I [29] are fully exchanged.…”

“…To exchange the buffer from H 2 Ot oD 2 O, complex Is amples were repeatedly washed with D 2 Ob uffer by ultrafiltration for at least 3h at 4 8Cp rior to the experiments. This time is necessary to ensure that the solvent-accessible, exchangeable water molecules bound to complex I [29] are fully exchanged.…”

Chiral Purity of Crystals Using Low‐Frequency Raman Spectroscopy

“…Recently, we reported on a new method for characterizing chirality of crystals based on the use of low‐frequency (LF) Raman spectroscopy . Organic crystalline structures have vibrational modes associated with covalent chemical bonds, intermolecular interactions, and overall oscillations of the crystal lattice ,. The development in recent years of volume holographic gratings (VHG) enabled a more facile use of Raman spectroscopy for the detection of low‐frequency vibrational (LFV) modes ,.…”

Specific Far Infrared Spectroscopic Properties of Phospholipids

“…Importantly, functional groups involved in hydrogen bonding have IR absorption bands that are temperature and pressure dependent [9][10][11]. Different specific spectral regions can be distinguished, and so-called IR continua have been described involving hydrogen bonding features [12][13][14][15]. In the far infrared (FIR) region, this continuum is seen at about 400 to 0 cm −1 , the exact position and broadness depending on the polarizability of the hydrogen bonding features.…”