Electrochemistry of Biomimetic Membranes - Applications of Electrochemistry and Nanotechnology in Biology and Medicine - page 250

with microporous biobeads, the tethered proteins are surrounded
by lipid molecules that form a lipid bilayer around them, as veri-
fied by SPR and EIS; a water layer remains interposed between the
lipid bilayer and the NTA moiety, acting as an ionic reservoir.
This approach has been adopted to investigate the function of
cytochrome c oxidase (COX) from the proteobacterium Rhodobac-
ter sphaeroides . 237 COX is the last enzyme in the respiratory elec-
tron transport chain of bacteria, located in the bacterial inner
membrane. It receives one electron from each of four ferro-
cytochrome c molecules, located on the periplasmic side of the
membrane, and transfers them to one oxygen molecule, converting
it into two water molecules. In the process, it binds four protons
from the cytoplasm to make water, and in addition translocates
four protons from the cytoplasm to the periplasm, to establish a
proton electrochemical potential difference across the membrane.
In this protein-tethered bilayer lipid membrane (ptBLM), the
orientation of the protein with respect to the membrane normal
depends on the location of the histidine stretch (his tag) within the
protein. Two opposite orientations of the protein were investigat-
ed, either with the cytochrome c binding side pointing away from
the electrode surface or directed toward the electrode, simply by
engineering the his tag on the C terminus of subunit SU I or SU II,
respectively. The individual steps of functionalization of the gold
support, adsorption of the engineered protein and its reconstitution
in the lipid bilayer were followed in situ by means of surface-
enhanced infrared absorption spectroscopy (SEIRAS). 237 The func-
tional activity of COX was verified by cyclic voltammetry with
both protein orientations. In this connection, it should be noted that
electron transfer in COX occurs sequentially through the four re-
dox centers Cu A , heme a, heme a 3 and Cu B , in the direction from
the binding site of cytochrome c , located on the outer side of the
bacterial membrane, to its inner side. With the cytochrome c bind-
ing side pointing away from the electrode surface, the primary
electron acceptor, Cu A , is far from the electrode surface. Hence, in
the absence of cytochrome c , the cyclic voltammogarm exhibits
only a capacitive current. This indicates that COX is not electrical-
ly coupled to the electrode, and direct electron transfer does not
take place. Under these conditions, the electrochemical impedance
spectra were fitted to an equivalent circuit consisting of a capaci-
tance C s , with in series an R m C m mesh and the solution resistance