Initially, transmembrane ET experiments with AM1 were performed using the ITS / K3Fe(CN)6
system introduced in Section 4.2.2. AM1 proteoliposomes were produced through the detergent depletion procedure described in Appendix A1.7.3; the only change to this procedure was the addition of 30
mM K3Fe(CN)6. One stoichiometric equivalent of heme was added to SDS-solubilized AM1
prior to detergent removal. The proteoliposomes were sonicated, extruded, and passed through a PD-10 column equilibrated to an isotonic Tris / KCl buffer solution to remove non-encapsulated K3Fe(CN)6. Finally, an additional three equivalents of heme were added prior to deoxygenation
and stop-flow mixing with reduced ITS. Unfortunately the measured rate of ITS oxidation in this system did not consistently increase between holo-AM1 and heme alone (similar to AP6 experiments in Figure 5.6, data not shown).
It is possible that the failure to observe robust transmembrane ET arose not from inherent “ET-incompetence” in AP6 or AM1, but instead was a feature of other components in the experimental system. Perhaps ITS or ferricyanide do not couple well for ET with the maquette hemes. To test this notion, the AM1 vesicle experiment was repeated, replacing the ITS and
Figure 5.9. AM1 heme reduction by FMN. (A) Q-band absorbance changes after continuously illuminated stopped-
flow mixture of oxidized cyt c and
vesicles encapsulating FMN and
EDTA. (B): Q-band absorbance
changes in detergent-solubilized AM1
binding 3 heme equivalents after (red)
2 min continuous illumination with FMN
and EDTA and (black) reacting with
excess dithionite. -0.02 0 0.02 0.04 0.06 0.08 520 540 560 580 Δ OD Wavelenth (nm) FMN+EDTA Dithionite -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 500 520 540 560 580 600 Δ OD Wavelength (nm) e- FMN + EDTA
Cyt c Heme Only
Detergent AM1+Heme
A
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K3Fe(CN)6 with other soluble redox reagents. A mixture of 8.5 mM free flavin mononucleotide
(FMN) and 28 mM EDTA replaced the K3Fe(CN)6 inside the vesicles. 35 µM cyt c replaced the
ITS in the surrounding solution (See small cartoon, Figure 5.9-A). Note that bound flavin cofactors, in conjunction with EDTA in solution, have been used as photoreductants in previous maquette designs[32], [25]. The system described here replaces a maquette-bound flavin with soluble, unbound FMN.
There are several significant experimental differences between these two systems. First, FMN is a photoactive species, requiring illumination in the stopped flow to drive ET. Upon excitation, the flavin becomes strongly oxidizing, with an Em on the order of +2.0 V. Excess
EDTA serves as an irreversible “sacrificial” electron donor to quench the excited flavin, producing a singly-reduced radical semiquinone state. This process may be repeated to yield a doubly-reduced FMN. At pH 8.0 the Em for the first electron in the doubly-reduced FMN is
about -260 mV at pH 8.0[33]; this is a sufficiently reducing to drive the initial maquette heme reduction. Second, electrons are directed inward to encapsulated oxidant in the ITS / K3Fe(CN)6
system, while they move outward in the FMN / cyt c system. A third difference in this arrangement is that the spectroscopically active species (cyt c) lies at the end of the redox chain, so cyt c redox change should only be observed upon complete transmembrane ET. In contrast, the ITS donor in the previous system lies at the beginning of the redox chain and thus could report oxidation even if ET failed to reach all maquette-bound hemes or the terminal acceptor.
The continuous light source employed to drive ET from the FMN largely ‘blinded’ the PMTs in the stopped-flow spectrometer, so it was not practical to track the spectral changes continuously after mixing. Instead, the light was periodically turned off to acquire “snapshots” over the course of the reaction. Figure 5.9-A shows a typical result after 120 sec illumination. The presence of heme, both with and without AM1, shows similar reduction of cyt c as indicated by the development of the reduced heme C α-band at 550 nm. AM1 is not helping to mediate cyt c reduction here. All the cyt c was quickly reduced upon lysing the liposomes with octyl-POE detergent, allowing direct contact between FMN and cyt c.
As a final control, I tested the ability of the photoexcited FMN, with excess EDTA as a sacrificial electron donor, to reduce heme in detergent-solubilized AM1; no lipid vesicles or cyt c
were present in this experiment. 10 µM AM1 with three heme equivalents in 2 mM DDM was combined with 300 µM FMN and 5.4 mM EDTA and illuminated for 2 min. Excess dithionite was added to reduce any remaining oxidized heme. Figure 5.9-B shows the results of this
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experiment; the peak at 560 nm is the reduced B-heme α-band. As expected, all heme was
reduced by dithionite. FMN reduced only about half the heme in this system. This is a surprising result, suggesting that the potential of the activated FMN is insufficient to reduce all the hemes in AM1. Potentiometric measurements of obligate single-heme AM1 variants could help to address this possibility. Alternately, perhaps the small dithionite ion (S2O42-) is able to access all AM1
hemes whereas the larger flavin is sterically occluded from some population.
Replacing AP6 with AM1 still did not produce a robust maquette-mediated transmembrane ET system. The next chapter discusses possible explanations in the context of Moser-Dutton Ruler calculations.