Abstract
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.
Highlights
Electron transfer (ET) is one of the principal processes involved in harvesting and transferring energy in natural and artificial systems
Small-Angle X-ray Scattering (SAXS) shows that the thicknesses of the liposomes,[62] is insignificantly altered when either of the probes resides in the membrane
Introduction of the spin-label at different positions on the fatty-acid chain permitted construction of a molecular “ruler” for monitoring ET in natural and artificial membrane systems by precise control of the donor/acceptor distance. We have used this experimental system to determine both the rate constants and activation energies for ET between [Ru(bpy)3]3+ and the nitroxide moieties in 5-doxyl stearic acid (5DSA) and 16-doxyl stearic acid (16DSA) embedded in unilamellar DMPC liposomes
Summary
Electron transfer (ET) is one of the principal processes involved in harvesting and transferring energy in natural and artificial systems. This method provides the depth parameter Φ (see the Experimental Section, and Supporting Information Figure S8 and Table S2), which is a measure of the immersion depth of the spin label.[61] Φ values of 1.5 and 2.9 were obtained for 5DSA and 16DSA, respectively These values are very similar to those reported for structurally related 5DPC (1-palmitoyl-2-stearoyl-(5doxyl)-sn-glycero-3-phosphocholine, Φ = 1.4) and 14DPC (1-palmitoyl-2-stearoyl-(14-doxyl)-sn-glycero-3-phosphocholine, Φ = 3.0) in comparable POPC membranes (Table S2).[68] This strengthens our contention that the nitroxide moiety in 16DSA is positioned substantially deeper within the bilayer than 5DSA, and that back-folding does not make a substantial contribution, in agreement with earlier findings.[69]. As proved by the lineshapes of the cw-EPR spectra and by the power saturation EPR experiments on symmetric liposomes, 5DSA and 16DSA occupy markedly distinct positions in the bilayer and should give rise to different electron transfer rates Following these observations, we solved the kinetic scheme and fitted the different time traces. Involvement of a hopping mechanism appears to be unlikely, since no electroactive component (e.g., an aromatic group) is located between the donor and the acceptor.[79]
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