Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.     

An electrochemical approach of the redox behavior of water insoluble ubiquinones or plastoquinones incorporated in supported phospholipid layers

Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)'s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

The influence of structure and redox state of prenylquinones on thermotropic phase behaviour of phospholipids in model membranes

Our study was aimed to investigate the significance of the isoprenoid side chain size as well as redox state of the quinone ring for interaction of two main classes of prenylquinones: plastoquinones (PQ) and ubiquinones (UQ) with lipid bilayers. By use of differential scanning calorimetry (DSC) we have followed the thermotropic behaviour of multilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC) upon incorporation of increasing amount (1.3-12 mol%) of quinone (quinol) molecules. Our studies reveal that as the side chain is shorter (from 9 to 2 isoprenoid units) the height of the calorimetric profiles is reduced and the temperature of the main transition of DPPC (T(m)) decreases (T(m)=39.4 degrees C for a sample with 12 mol% of PQ-2), and then increases up to 39.8 degrees C for PQ-1. For the samples containing quinols the effect is more pronounced even at lower concentration. The greater influence of the added prenylquinones on the pretransition demonstrates a stronger distortion of the DPPC packing in the gel state. It seems that this is the isoprenoid side chain length rather than the redox state of prenylquinones that determines their effectiveness in perturbation of thermotropic properties of lipid bilayer.     

Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

An approach to evaluate two-electron reduction of 9,10-phenanthraquinone and redox activity of the hydroquinone associated with oxidative stress

Quinones are widely used as medicines or redox agents. The chemical properties are based on the reactions against an electron donor. 9,10-Phenanthraquinone (PQ), which is a quinone contaminated in airborne particulate matters, forms redox cycling, not Michael addition, with electron donors. Redox cycling of PQ contributes to its toxicity, following generation of reactive oxygen species (ROS). Detoxification of quinones is generally thought to be two-electron reduction forming hydroquinones. However, a hydroquinone of PQ, 9,10-dihydroxyphenanthrene (PQH(2)), has been never detected itself, because it is quite unstable. In this paper, we succeeded in detecting PQH(2) as its stable derivative, 9,10-diacetoxyphenanthrene (DAP). However, higher concentrations of PQ (>4 microM) form disproportionately with PQH(2), producing the 9,10-phenanthraquinone radical (PQ(-)) which is a one-electron reducing product of PQ. In cellular experiments using DAP as a precursor of PQH(2), it was shown that PQH(2) plays a critical role in the oxidative protein damage and cellular toxicity of PQ, showing that two-electron reduction of PQ can also initiate redox cycling to cause oxidative stress-dependent cytotoxicity.     

Analysis of the differences in microbial community structures between suspended and sessile microorganisms in rivers based on quinone profile

In this study, a quinone profiling method was applied to clarify the differences in community structure between suspended and sessile microorganisms in rivers. The compositions of microbial quinone of 6 sites for 4 rivers were analyzed. Ubiquinone (UQ)-8, UQ-10, menaquinone (MK)-7, and plastoquinone (PQ)-9 were observed in all samples of suspended and sessile microorganisms for the sites investigated. The dominant quinone species in suspended microorganisms was ubiquinone, and that in sessile microorganism was photosynthetic quinones (namely PQ-9 and vitamin K1). This indicated that aerobic bacteria were abundant in the suspended microorganisms, and photosynthetic microorganisms such as micro-algae and cyanobacteria dominated in the sessile microorganisms. The quinone concentration in the river waters tested, which reflects the concentration of suspended microorganisms, ranged from 0.045 to 1.813 nmol/L. The microbial diversities of suspended and sessile microorganisms calculated based on the composition of all quinones were in the range from 3.4 to 7.5, which was lower than those for activated sludge and soils. Moreover, the diversity of heterotrophic bacteria for sessile microorganisms in the rivers was higher than that for the suspended microorganisms.     

Effect of quinones on the photoelectric properties of chlorophylla-containing lipid bilayers

Summary A Mueller-Rudin lipid bilayer formed from egg lecithin, cholesteroln-decane, and chlorophylla when separating asymmetric solutions of oxidant and reductant is capable of developing both a photoconductance and a photovoltage when exposed to continuous light focused on the bilayer region. The incorporation of plastoquinones (PQ-5 or PQ-9) increases the dark and photoconductances up to twenty times. Other quinones and hydroquinones also increased the conductance. The role of pH gradients on the electrical conductivity is discussed in terms of proton and electron transfer across the membrane solution interfaces.     

Quinone interactions with the chloroplast cytochrome b6-f complex

The requirements for reconstitution of electron transfer activity with a plastoquinone (PQ)-depleted cytochrome b6-f complex from spinach have been considered. Full restoration of activity measured as plastocyanin reduction with either duroquinol in the dark or Photosystem II (PSII) in the light requires both PQ-9 and phospholipid. However, a substantial dark activity can be observed with duroquinol and phospholipid in the absence of any added PQ-9. PSII, with its associated PQ molecules, can also donate electrons in the light to the cytochrome complex which has been depleted of plastoquinone. Electron donation by duroquinol in the dark to the PQ-depleted cytochrome complex is stimulated by PSII, and this stimulation is dependent on the presence of the two PQ molecules in the PSII preparation. Measurements of proton translocation with the PQ-depleted complex indicate this quinone is not required for the observed H+/e- ratio of 2. Studies of cytochrome b6 kinetics with the free and liposome-incorporated PQ-depleted complex show this cytochrome undergoes redox reactions similar to those of a control complex which contains PQ. These results indicate the PQ that copurifies with the cytochrome complex is not essential for any of the measured activities. These findings are considered in relation to a quinone binding site(s) in the cytochrome complex which is not specific to PQ but can bind other quinones, such as duroquinol, in a lipid-dependent process.     

The electrical and spectroscopic properties of planar asymmetrical membranes incorporating chlorophyll a and plastoquinone-9. Model of incorporation of chlorophyll a and plastoquinone-9

We have studied the incorporation of chlorophyll a and plastoquinone-9 in Montal-Mueller membranes. In particular, we have been interested by the influence of both the lipid : chlorophyll a ratio and the asymmetry of incorporation of the constituents on the electrical and fluorescence spectroscopic properties of the planar membranes built up from these constituents. The phospholipid matrix was made from phosphatidylethanolamine and phosphatidylserine. The monitoring of the fluorescence spectral properties of chlorophyll a incorporated in various concentrations leads to the conclusion that chlorophyll a is incorporated in the bilayers in monomeric form inside microdomains. It is shown that chlorophyll a is positioned in these microdomains in such a way that the porphyric ring is interacting with the polar head of the lipid molecules where the interface polarity shows a dielectric constant varying between 25 and 35. The phytyl chain is embedded in the bilayer core, serving as an anchor, running parallel to the aliphatic chains of the phospholipids. We have also monitored the position of the plastoquinone-9 molecules within the bilayer. We found that plastoquinone-9 is incorporated in the center plane of the bilayer, increasing the thickness of the bilayer. This result confirms evidence, gathered in the literature from monolayer and differential scanning calorimetry studies, that long chain quinones and especially plastoquinone-9 are embedded deeply within the hydrophobic core of the bilayer. We also show that when chlorophyll a and plastoquinone-9 are present together in the bilayer, the quinolic ring of the plastoquinone-9 molecule positions itself in the free volume created by the bulky porphyric ring of a chlorophyll a molecule.     

Plastoquinone B

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K₁ (10, 12), an unknown naphthoquinone (13) and α-, β- and γ-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R_F values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.     

Hydrophobic mismatch in gramicidin A'/lecithin systems.

Gramicidin A' (GA') has been added to three lipid systems of varying hydrophobic thicknesses: dimyristoyllecithin (DML), dipalmitoyllecithin (DPL), and distearoyllecithin (DSL). The similarity in length between the hydrophobic portion of GA' and the hydrocarbon chains of the lipid bilayers has been studied by using 31P and 2H NMR. Hydrophobic mismatch has been found to be most severe in the DML bilayer system and minimal in the case of DSL. In addition, the effects of hydrophobic mismatch on the cooperative properties of the bilayer have been obtained from 2H NMR relaxation measurements. The results indicate that incorporation of the peptide into the bilayer disrupts the cooperative director fluctuations characteristic of pure multilamellar lipid dispersions. Finally, the GA'/lecithin ratio at which the well-known transformation from bilayer to reverse hexagonal (HII) phase occurs (Van Echteld et al., 1982; Chupin et al., 1987) is shown to depend on the acyl chain length of the phospholipid. A rationale is proposed for this chain length dependence.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH >11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH >9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q(A)Q(B)(-) state is stabilized by about 40 meV at 6.511. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and (31)P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

Biophysical studies on agents affecting the state of membrane lipids: Biochemical and pharmacological implications

Summary The phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins.We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids.The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of -helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition.The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics.The conformational effect of anesthetics is not directly exerted on the porteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition.     

Structures of mammalian cytosolic quinone reductases

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.     

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ(9)) and rhodoquinone-9 (RQ(9)) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ(2)) and ubiquinone-2 (UQ(2)) reduction. Structure-activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ(2) and NADH-UQ(2) oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ(2) and UQ(2), respectively. The pH profiles of both activities differed remarkably; NADH-RQ(2) oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ(2) oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ(2) and UQ(2) as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.     

Incorporation of the V-ATPase inhibitors concanamycin and indole pentadiene in lipid membranes. Spin-label EPR studies

The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase.     

Synthetic quinones influencing herbicide binding and Photosystem II electron transport. The effects of triazine-resistance on quinone binding properties in thylakoid membranes

We have analyzed the binding of synthetic quinones and herbicides which inhibit electron transport at the acceptor side of Photosystem II (PS II) of the photosynthetic electron-transport chain in thylakoid membranes. These data show that quinones and PS II-directed herbicides compete for binding to a common binding environment within a PS II region which functions as the Q⁻ / PQ oxidoreductase. We observed that (1) synthetic quinones cause a parallel inhibition of electron transport and [¹⁴C]herbicide displacement, and (2) herbicide binding is affected both by the fully oxidized and fully reduced form of a quinone. Quinone function and inhibitor binding were also investigated in thylakoids isolated from triazine-resistant weed biotypes. We conclude the following. (1) The affinity of the secondary accepting quinone, B, is decreased in resistant thylakoids. (2) The observation that the equilibrium concentration of reduced Q after transferring one electron to the acceptor side of PS II is increased in resistant as compared to susceptible chloroplasts may be explained both by a decrease in the affinity of PQ for the herbicide / quinone binding environment, and by a decrease of the midpont redox potential of the B / B⁻ couple. (3) The binding environment regulating quinone and herbicide affinity may be divided roughly into two domains; we suggest that the domain regulating quinone head-group binding is little changed in resistant membranes, whereas the domain-regulating quinone side-group binding (and atrazine) is altered. This results in increased inhibitory activity of tetrachloro-p-benzoquinone and phenolic herbicides, which are hypothesized to utilize the quinone head-group domain. The two domains appear to be spatially overlapping because efficient atrazine displacement by tetrachloro-p-benzoquinone is observed.     

Composition and function of plastoglobuli

The lipid composition of whole leaves and isolated plastoglobul of beech (Fagus sylvatica) has been studied during four natural autumnal senescence stages. Chlorophylls, glycolipids, and phospholipids were extensively degraded in leaves. About 20% of the glycolipids found in leaves during summer, however, remained in the last stage of leaf senescence. Triacylglycerols, also detected in large amounts in summer leaves, were hydrolyzed during senescence. The content of free fatty acids derived from degradation of glycerolipids therefore increased. The total carotenoid and prenyl quinone content was largely unchanged during senescence, except during the last stage investigated, but the reduced forms of prenyl quinones decreased while the oxidized prenyl quinones increased. Plastoglobuli isolated from summer leaves mainly contained triacylglycerols, plastohydroquinone, and -tocopherol. The triacylglycerol content declined in plastoglobuli during senescence. Most of the triacylglycerols must be located outside the plastoglobuli throughout the stages investigated. Carotenoids liberated from thylakoids were esterified and increasingly deposited in plastoglobuli during senescence. In the last senescence stage, carotenoid esters were the main component of plastoglobuli. Prenyl quinones were also transferred into plastoglobuli. Reduced prenyl quinones were sucessively oxidized during senescence and plastoquinone (oxidized) was the predominant prenyl quinone in plastoglobuli isolated from the last senescence stage. The carotenoid and prenyl quinone distribution was identical in leaves and plastoglobuli during late senescence. The main constituents of thylakoids, glycolipids and proteins, were not deposited in plastoglobuli and therefore did not play an important role in plastoglobuli metabolism.Abbreviation PQ plastoquinone     

The role of phospholipids in the reduction of ubiquinone analogues by the mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase complex.

The isolated NADH-ubiquinone oxidoreductase complex of bovine heart mitochondria reduces ubiquinone analogues by two pathways. One pathway is inhibited by rotenone, and reduction of quinones takes place in the lipid phase of the system. The other pathway is insensitive to rotenone and reduction takes place in the aqueous phase. The variation of rates of electron transpport with the chemical nature of the quinone analogue and the concentrations of both quinone and phospholipid can be rationalized in terms of partition of the quinone between the aqueous and lipid phases of the system. Thus one function of phospholipid associated with the enzyme appears to be to act as solvent for ubiquinone reduced by the rotenone-sensitive pathway. This proposal is supported by the kinetic behaviour of enzyme whose endogenous lipids have been replaced by (1,2)-dimyristoylsn-glycero-3-phosphocholine. Thus, under certain circumstances, the rotenone-sensitive reduction of ubiquinone-1 exhibited a substantial increase in activation energy below the phase-transition temperature of the synthetic lipid, whereas the reduction of other acceptors was unaffected.