SFB 1078/1: THz spectra of protein surface regions depending on the catalytic state (TP A 03)


Proton-uptake from the cytoplasmatic side of Cytochrome c oxidase (CcO) has an apparent second-order rate constant which exceeds the diffusion-controlled rate in the bulk by an order-of-magnitude. This discrepancy led to the concept of a “proton-collecting antenna” around each channel entrance, consisting of negatively charged residues with overlapping Coulomb cages and protonable residues. They capture protons collectively and add their (first-order) rates-of-transfer to the reaction center. Whereas proton diffusion can be measured with fluorescent pH probes, the underlying structural fluctuations of the hydrated protein surface have not yet been observed. Here we use electric absorption spectroscopy in the far-infrared and terahertz (THz) range 0.1-200 cm-1 to characterize the proton-collecting antenna together with its hydration layer. At low frequencies, intermolecular fluctuations of polar residues and solvation water are observed directly (in contrast to marker bands > 1000 cm-1, due to intramolecular vibrations which only depend on H-bonding). Overwhelming absorption by bulk water is avoided with molecular THz probes, small betains which are linked to specific Cys sites. Regioselectivity is made possible by an effective observation radius of 15 Å. We will first record spectra in the fully oxidized and reduced states of CcO, for different sites and at various pH 7.5. These will be correlated with the apparent proton diffusion constant under the same conditions. Later such THz spectra may be recorded during the catalytic cycle and understood with the help of full-scale Molecular Dynamics simulations. Measurements of proton uptake/release (TP A2) will be supported by special fluorescent pH indicator dyes which are similarly linked to target Cys residues.


Projektleitung
Ernsting, Nikolaus Prof. Ph. D. (Details) (Physikalische und Theoretische Chemie II)

Mittelgeber
DFG: Sonderforschungsbereich - Beteiligungen

Laufzeit
Projektstart: 01/2013
Projektende: 12/2016

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Zuletzt aktualisiert 2022-09-09 um 01:05