Abstract:
Cytochrome c oxidase .inserted into proteoliposomes
translocates protons with a stoichiometry of approx-,
imately 0.4-0.6 H+/e- in the presence of valinomycin
plus pottasium. The existance .ofsuchproton translocation
is .supportedby experiments with lauryl maltoside
which abolished the pulses but~~d not inhibit cyt.
c binding .or oxidase turnover. Pulses with K3FeCN6 did
not induce acidification further supporting vectorial
proton transport by cyt ..aa3 . Upon lowering the ionic
strength and pulsing with ferrocytochrome c, H+/eratios
increased. This increase is attributed to scaler
proton release consequent upon cyt.c-phospholipid binding.
Oxygen pulses at low ionic strength however did not exhibit
this large scaler increase in H+/e- ratios.A-small increase
was observed upon .02 pul'sing at·low ionic strengt.h. This
increase was KeN and, ,pcep sensitive and thus possibly due
to a redox linked scaler deprotonation. Increases in the
H+/e- ratio also occurred ifp~lses ,were performed in the
presence of nonactin rather.than valinomycin.
The fluorescent pH indicator pyranine was internally
trapped inaa3 conta~ning "proteoliposomes. Internal alkalinization,
as mon,itored by pyranine fluorescence leads
to a of approx.imately 0.35 units, which is proportional
to electron flux. This internal alkalinization was also
DCCD sensitive, being inhibited by approximately 50%. This
50% inhibition of internal alkalinization supports the existance of vectorial proton transport.
