Biological Electron Transfer Mediators in Model Membrane Systems

Lyle, Ian G (1978) Biological Electron Transfer Mediators in Model Membrane Systems. PhD thesis, University of Glasgow.

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The overall objective of this research was to create chemically well-defined liquid redox membranes, each of which would be capable of mediating a process of continuous electron transfer between an aqueous reductant on one side and an aqueous oxidant on the other. The transport of electrons between the two otherwise separate aqueous phases would be effected by membrane-bound reversible redox-active carrier molecules. It has been proposed that such molecules function in certain electron transfer processes associated with biological membranes. In particular, the isoprenoid benzoquinone coenzyme Q is thought to carry electrons (and protons) across the inner mitochondrial membrane during respiration, whilst the related naphthoquinone vitamin K acts as a carrier in photosynthetic and bacterial electron transfer. Since these two molecules were also found to possess all the attributes required of 'ideal' carriers, they were chosen for incorporation in all the redox membranes investigated during this research, in the hope that their behaviour in these simple model systems could be used to elucidate their biological function. Preliminary investigations were carried out to find suitable aqueous reductants and reducible substrates. A wide variety of common redox reagents, and several biological molecules, were tested for their ability to reduce coenzyme Q10 and vitamin K1, or to reoxidise their quinol forms, in biphasic reactions in which the quinone/quinol in hexane solution was equilibrated anaerobically over the aqueous reagent. A number of criteria were specified defining 'ideal' reductants, substrates and carriers, and control experiments were performed to assess the ideality of the reagents used. In general terms, coenzyme Q was found to be reduced by a wider range of reductants than vitamin K, while dihydrovitamin K was much more easily reoxidised than reduced coenzyme Q. Both quinones reacted readily at pH 7 with reduced methyl viologen and flavin mononucleotide free radicals, which behaved as ideal reductants. Electron paramagnetic resonance spectroscopy revealed the presence of semiquinone free radical species of coenzyme Q and vitamin K at the hexane/aqueous interface during these reactions. To establish continuous membrane-mediated electron transfer processes, experiments were conducted in an H-shaped reaction vessel in which the quinone solution (in hexane) formed a bridge between the aqueous reductant and aqueous oxidant in the lower limbs. Reduced methyl viologen, in large molar excess over the quinone and substrate, was routinely used as a reductant, and the kinetics of reduction of various substrates were followed spectrophotometrically. Both coenzyme Q and vitamin K were shown to function as reversible electron carriers in these bulk membrane systems, but the rate of substrate reduction was always very much slower when coenzyme Q was used. For this reason, more detailed studies were restricted to vitamin K as electron carrier. The kinetics of reduction of methylene blue by dihydrovitamin K were first order with respect to the oxidised dye, and the measured rate constant was consistent with diffusion control on the substrate side of the interface. The reaction was inhibited to varying degrees by the addition of different amphipathic phospholipids to the membrane. With cytocnrome c as substrate, the biphasic reaction with dihydrovitamin K was no longer diffusion-controlled, but was determined by mechanistic factors. The very slow, apparently zero-order reaction was greatly stimulated by the addition of the mitochondrial phospholipid cardio-lipin to the membrane, and the variation of the reaction rate with ionic strength of the aqueous phase could be explained in terms of binding between the acidic phospholipid and the basic protein. Such interactions are proposed to be of importance in the functional organisation of the mitochondrial membrane. Having established the abilities of coenzyme Q and vitamin K to act as electron carriers across a bulk hydrocarbon phase, attempts were made to improve the biological model by reducing the thickness of the membrane to the dimensions of a lipid bilayer. The two model systems studied were planar bimolecular lipid membranes (BLM) and closed unilamellar lipid vesicles (liposomes). The stabilities and thicknesses of a large number of BLM formed from a selection of amphipathic lipids and lipid mixtures were examined, and 'recipes' were found for membrane-forming solutions which yielded stable bilayers containing either vitamin K or coenzyme Q. A membrane cell was developed, allowing electron transfer across ultrathin membranes to be followed spectrophotometrically. Unfortunately, the instability of lipid bilayers within the apparatus did not allow kinetics experiments to be performed using BLM. However, electron transfer across thicker lens membranes, mediated by vitamin K, was demonstrated. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Chemical engineering, Bioengineering
Date of Award: 1978
Depositing User: Enlighten Team
Unique ID: glathesis:1978-78783
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 14:54
Last Modified: 30 Jan 2020 14:54

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