In principle, one would expect significant protein-mediated electron transfer across a membrane using either AP6 or AM1. This section introduces elementary Moser-Dutton Ruler computations to predict electron transfers between transmembrane redox centers, and discusses possible reasons why these rates have not been observed experimentally. The Moser-Dutton Ruler, introduced in Section 1.2.2, is an empirical relation derived from measured intra- and inter-protein electron transfer rates in a variety of natural and modified proteins:
Eqn. 1.5
where kET is electron transfer rate in s-1, R is the edge-to-edge cofactor distance in Å, and ΔG and
λ are the free energy change and reorganization energy in eV. In the following analysis we
extend the Moser-Dutton Ruler to also include electron transfers between AP6/AM1 terminal hemes and soluble redox species (ITS, K3Fe(CN)6, FMN, or cyt c) on opposite sides of the
membrane. Inter-heme distances were estimated by assuming straight helices and measuring edge-to-edge distances between cofactors placed in Pymol models. ET distances involving soluble molecules were estimated by adding 3 Å to the closest solvent approaching the appropriate cofactor. The set of electron transfer rates between all cofactors yields a system of ordinary differential equations amenable to numerical solution. A Python program computed ET
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rates and solved the ODEs for AP6 and AM1. Importantly, there are two possible vectorial orientations for the amphiphilic maquettes with respect to the membrane; the aqueous region of the protein can extend either into the interior or exterior of the vesicle. In certain specific cases, the selection of lipid composition can control vectorial orientation of reconstituted membrane protein, but typically the orientation is random and should be assumed to be a combination of the two possibilities [34]. Since these two vectorial arrangements differ in their predicted ET rates, the system was solved for both orientations in AP6 and AM1. These models assume that maquette hemes in the membrane and aqueous regions use the higher and lower measured Em
values at pH 8.0. Em values for soluble ITS, K3Fe(CN)6, FMN, and cyt c are, respectively, taken
as -90 mV, 420 mV, -260 mV[33], and 250 mV. Figure 5.10 shows these results.
The calculations do not account for collisions with the soluble species. Instead, they make the major simplification that the soluble donor and acceptor are stationary. However, the models may reasonably be used to make qualitative comparisons between the systems, particularly in the time needed for an electron to traverse the membrane and reduce the soluble acceptor species. Table 5.5 shows the computed terminal reduction half-times. Interestingly, the first, third, and fourth rows show very similar times. Only the “aqueous-in” orientation of AP6 is predicted to be significantly slower.
Maquette Protein Orientation w/r/t vesicle
t1/2 (s)
AP6 Aqueous region out 7.2 x 10-4
AP6 Aqueous region in 3.1 x 10-3
AM1 Aqueous region out 6.0 x 10-4
AM1 Aqueous region in 7.3 x 10-4
Table 5.5 Computed half-times for transmembrane ET.
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AP6 offers six heme-binding sites, and these are located at three distinct positions along the length of the tetrahelical bundle (i.e. ligated at one of three histidines on the single-helix monomer). AP6 binds 4 hemes with at least 1 µM affinity (Table 4.2), although there is minimal binding on the remaining sites. If the four hemes are bound such that one position lacks any hemes, the system would lack a complete ET chain and transmembrane ET would be suppressed. AM1, with only three heme sites, has a liposome Kd of 4 µM at its third site. Although not
sufficient to ensure complete occupation for 3.5 AM1 µM concentrations employed in these
Figure 5.10: AP6 and AM1 Moser-Dutton ET calculations. Each plot is computed by numerically solving the system of differential equations for ET, given Moser-Dutton rates. The glyph on each plot represents the arrangement of redox centers across the membrane (yellow band). Green, red, and blue rectangles are maquette bound hemes, while blue and purple rectangle denote redox-active species in solution. The colored traces give fraction of each redox center predicted to be reduced. The model assumes that at t=0, the only reduced species
is the soluble electron donor (ITS of FMN). A) AP6 proteoliposomes with K3Fe(CN)6 inside the
vesicles and initially-reduced ITS outside. The maquette is oriented such that the aqueous
heme region faces the exterior of the lipid vesicle, interacting with aqueous ITS. B) As frame
A), except that the maquette is oriented with the aqueous region facing the vesicle interior. C)
AM1 proteoliposomes with photo-reduced FMN inside and oxidized cyt c outside. The aqueous
region faces outward. D) As C), except that maquette is oriented with the aqueous region
facing the vesicle interior.
1 2 5 6 1.0 0.8 0.6 0.4 0.2 0.0 F ra ct io na l Po pu la tio n 1.0 0.8 0.6 0.4 0.2 0.0 F ra ct io na l Po pu la tio n
Log10 Time [s] Log10 Time [s]
A B D C Cyt c FMN Cyt c FMN ITS FeCN ITS FeCN
FMN=-260
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experiments, there should be enough fully-heme-equipped maquettes to exhibit significant ET if the system is in fact capable of heme-mediated ET across the bilayer.
The absence of a 560 nm peak in Figure 5.9-A shows that even after 2 minutes of illumination in flavin-encapsulating proteoliposomes, there is no persistent reduction of maquette-bound hemes in AM1. This suggests the failure may lie in the initial ET from soluble donor to the maquette. However, there is partial heme reduction when the same protein is solubilized in detergent (Figure 5.9-B). Most of this reduction is probably in the aqueous-region heme, as this cofactor would have closer access to the FMN donor. The lack of any discernable B-heme reduction in Figure 5.9-A thus suggests that #1) AM1 in vesicles is oriented predominantly with aqueous region facing outwards such that the acqueous region is on the opposite side of the membrane from FMN and #2) the initial electron transfer from FMN to the first membrane-bound heme is very slow or non-existent. Repeating the AM1 experiment with the FMN/EDTA donor system outside the vesicles could strengthen this conclusion.