Resonance-mode electrochemical impedance measurements of silicon dioxide supported lipid bilayer formation and ion channel mediated charge transport.

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).

Refractive-index-based screening of membrane-protein-mediated transfer across biological membranes.

Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore.

Self-assembly formation of multiple DNA-tethered lipid bilayers.

Inspired by natural cell-cell junctions, where membrane-residing proteins control the separation between two or more membranes without interfering with their integrity, we report a new self-assembly route for formation of multiple highly fluid tethered lipid bilayers with the inter-membrane volume geometrically confined by membrane-anchored DNA duplexes. The formation of multiple planar membrane-membrane junctions were accomplished using disk shaped bicelles, composed of a mixture of the long-chained dimyristoyl phosphatidylcholine (DMPC) and the short-chained dihexanoyl PC further stabilized with the positively charged detergent hexadecyl-trimethyl-ammonium bromide (CTAB). Quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) were used to monitor the formation and to characterize the integrity of the self-assembled lipid-DNA architecture.

Plasticity of proton pathway structure and water coordination in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

Localized proton microcircuits at the biological membrane-water interface.

Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters.

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.

Water-hydroxide exchange reactions at the catalytic site of heme-copper oxidases.

Membrane-bound heme-copper oxidases catalyze the reduction of O(2) to water. Part of the free energy associated with this process is used to pump protons across the membrane. The O(2) reduction reaction results in formation of high-pK(a) protonatable groups at the catalytic site. The free energy associated with protonation of these groups is used for proton pumping. One of these protonatable groups is OH(-), coordinated to the heme and Cu(B) at the catalytic site. Here we present results from EPR experiments on the Rhodobacter sphaeroides cytochrome c oxidase, which show that at high pH (9) approximately 50% of oxidized heme a(3) is hydroxide-ligated, while at low pH (6.5), no hydroxide is bound to heme a(3). The kinetics of hydroxide binding to heme a(3) were investigated after dissociation of CO from heme a(3) in the enzyme in which the heme a(3)-Cu(B) center was reduced while the remaining redox sites were oxidized. The dissociation of CO results in a decrease of the midpoint potential of heme a(3), which results in electron transfer (tau approximately equal 3 micros) from heme a(3) to heme a in approximately 100% of the enzyme population. At pH >7.5, the electron transfer is followed by proton release from a H(2)O molecule to the bulk solution (tau approximately equal 2 ms at pH 9). This reaction is also associated with absorbance changes of heme a(3), which on the basis of the results from the EPR experiments are attributed to formation of hydroxide-ligated heme a(3). The OH(-) bound to heme a(3) under equilibrium conditions at high pH is also formed transiently after O(2) reduction at low pH. It is proposed that the free energy associated with electron transfer to the binuclear center and protonation of this OH(-) upon reduction of the recently oxidized enzyme provides the driving force for the pumping of one proton.

The entry point of the K-proton-transfer pathway in cytochrome c oxidase.

Cytochrome c oxidase is a redox-driven proton pump. The enzyme has two proton input pathways, leading from the solution on the N-side to the binuclear center. One of these pathways, the K-pathway, is used for proton uptake upon reduction of the binuclear center. It is also important for local charge compensation during reaction of the fully reduced enzyme with O2. Two different locations have been proposed to constitute the entry point of the K-pathway: near S(I-299) or near E(II-101), respectively, in the Rhodobacter sphaeroides enzyme. The experiments discussed in this study are aimed at identifying the location of the entry point. The kinetics and extent of flash-induced proton release coupled to oxidation of heme a3 (tau congruent with 2 ms at pH 8.8 in the wild-type enzyme) in the absence of O2 were investigated in the ED(II-101), SD(I-299), and KM(I-362) mutant enzymes, i.e., at the two proposed entry points and in the middle of the pathway, respectively. This reaction was completely blocked in KM(I-362), while it was slowed by factors of 25 and 40 in the ED(II-101) and SD(I-299) mutant enzymes, respectively. During reaction of the fully reduced enzyme with O2, electron transfer from heme a to the catalytic site (during P(R)-formation) was blocked in the KM(I-362) and SD(I-299)/SG(I-299) but not in the ED(II-101)/ EA(II-101) mutant enzymes. The results are interpreted as follows: Residue K(I-362) is involved in both proton transfer and charge compensation (in different reaction steps). The impaired proton release in the S(I-299) mutant enzymes is an indirect effect due to an altered environment of K(I-362). E(II-101), on the other hand, is likely to be part of the K-pathway since mutation of this residue results in impaired proton release but does not affect the P(R) formation kinetics; i.e., the properties of K(I-362) are not altered. Consequently, we conclude that the entry point of the K-pathway is located near E(II-101).

The rate of internal heme-heme electron transfer in cytochrome C oxidase.

Cytochrome c oxidase catalyzes the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of dioxygen to water. The process involves a number of intramolecular electron-transfer reactions, one of which takes place between the two hemes of the enzyme, hemes a and a3, with a rate of approximately 3 x 10(5) s(-1) (tau approximately 3 micros). In a recent report [Verkhovsky et al. (2001) Biochim. Biophys. Acta 1506, 143-146], it was suggested that the 3 x 10(5) s(-1) electron transfer may be controlled by structural rearrangements and that there is an additional electron transfer that is several orders of magnitude faster. In the present study, we have reinvestigated the spectral changes occurring in the nanosecond and microsecond time frames after photolysis of CO from the fully reduced and mixed-valence enzymes. On the basis of the differences between them, we conclude that in the bovine enzyme the microscopic forward and reverse rate constants for the electron-transfer reactions from heme a to heme a3 are not faster than approximately 2 x 10(5) and approximately 1 x 10(5) s(-1), respectively.

Ligand binding reveals protonation events at the active site of cytochrome c oxidase; is the K-pathway used for the transfer of H(+) or OH(-)?

We have investigated the CO-recombination kinetics after flash photolysis of CO from the "half-reduced" cytochrome c oxidase as a function of pH. In addition, the reaction was investigated in mutant enzymes in which Lys(I-362) and Ser(I-299), located approximately in the middle of the K-pathway and near the enzyme surface, respectively, were modified. Laser-flash induced dissociation of CO is followed by rapid internal electron transfer from heme a(3) to a. At pH>7 this electron transfer is associated with proton release to the bulk solution (tau congruent with 1 ms at pH 8). Thus, the CO-recombination kinetics reflects protonation events at the catalytic site. In the wild-type enzyme, below pH approximately 7, the main component in the CO-recombination displayed a rate of approximately 20 s(-1). Above pH approximately 7, a slow CO-recombination component developed with a rate that decreased from approximately 8 s(-1) at pH 8 to approximately 1 s(-1) at pH 10. This slow component was not observed with KM(I-362), while with the SD(I-299)/SG(I-299) mutant enzymes at each pH it was slower than with the wild-type enzyme. The results are interpreted in terms of proton release from H(2)O in the catalytic site after CO dissociation, followed by OH(-) binding to the oxidized heme a(3). The CO-recombination kinetics is proposed to be determined by the protonation rate of OH(-) and not dissociation of OH(-), i.e. the K-pathway transfers protons and not OH(-). With the KM(I-362) mutant enzyme the proton is not released, i.e. OH(-) is not formed. With the SD(I-299)/SG(I-299) mutant enzymes the proton is released, but both the release and uptake are slowed by the mutations. During reaction of the reduced enzyme with O(2), the H(2)O at the binuclear center is most likely involved as a proton donor in the O-O cleavage reaction.