Systematic Quantification of Electron Transfer in
a Bare Phospholipid Membrane Using Nitroxide-Labeled Stearic Acids:
Distance Dependence, Kinetics, and Activation Parameters
posted on 2020-08-26, 01:13authored byMax Schmallegger, Antonio Barbon, Marco Bortolus, Angela Chemelli, Itzhak Bilkis, Georg Gescheidt, Lev Weiner
In
this report, we present a method to characterize the kinetics
of electron transfer across the bilayer of a unilamellar liposome
composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine.
The method utilizes synthetic phospholipids containing noninvasive
nitroxide spin labels having the >N–O• moiety at
well-defined
distances from the outer surface of the liposome to serve as reporters
for their local environment and, at the same time, permit measurement
of the kinetics of electron transfer. We used 5-doxyl and 16-doxyl
stearic acids. The paramagnetic >N–O• moiety is photo-oxidized
to the corresponding diamagnetic oxoammonium cation by a ruthenium
electron acceptor formed in the solution. Electron transfer is monitored
by three independent spectroscopic methods: by both steady-state and
time-resolved electron paramagnetic resonance and by optical spectroscopy.
These techniques allowed us to differentiate between the electron
transfer rates of nitroxides located in the outer leaflet of the phospholipid
bilayer and of those located in the inner leaflet. Measurement of
electron transfer rates as a function of temperature revealed a low-activation
barrier (ΔG‡ ∼ 40 kJ/mol) that supports
a tunneling mechanism.