Verlag des Forschungszentrums Jülich
JUEL-4009
Nyquist, Rebecca M.
Infrared Spectroscopy of Cytochrome C Oxidase Intermediate States
XIII, 178 S., 2003
Cytochrome c oxidase is a critical player in the process of cellular
respiration, performing proton translocation across a lipid bilayer, coupled to the
four-electron reduction of 02 to H2O. To accomplish this catalytic task,
specific changes at the active site influence chemical and physical changes
throughout the protein, altering amino acid side-chain orientations,
hydrogen bond lengths, and protonation states. Infrared spectroscopy is capable of
monitoring these changes. In this thesis work, cytochrome c oxidase was
specially prepared for perfusion-induced infrared difference spectroscopy. The
resulting infrared difference spectra demonstrate that the side-chain of a key
glutamic acid, E286 from Rhodobacter sphaeroides, is protonated in both
oxidized (0) and fully-reduced states with a pKa higher than 9.5. Also
presented in this work are the first infrared difference spectra for O2 bond-
cleaved intermediate states P and F. In addition, time-resolved infrared
spectroscopy was used to study vibrational differences between intermediate
states preceding 02 binding, the one- and two-electron reduced states (E
and R2, respectively). Taken together, the infrared difference spectra
presented here demonstrate that the E286 side-chain is deprotonated in E and
P but protonated in 0, R2, and F. This indicates that E286 transfers its
proton in the O→E and R2→P transitions; and that it accepts a proton in
the E→R2 and P→F transitions. Also, a tyrosine residue, presumably the
active site tyrosine Y288, was observed to be protonated in 0 and
deprotonated in F. These results spark interpretation of mechanistic models as well
as form the basis for future time-resolved infrared spectroscopic investigations.
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