Surface-coupled proton exchange of a membrane-bound proton acceptor

Abstract

Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pHsensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pK a s, revealed two distinct proton exchange regimes. At high pH (>8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (<7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.biomembrane | diffusion | electrostatic potential | fluorescence correlation spectroscopy (FCS) | proton transfer E nergy conversion in living cells typically involves proton translocation across a membrane, via proton transporters. These transporters maintain a proton electrochemical gradient utilizing free energy provided, for example by electron transfer or light. The free energy stored in this gradient is used, e.g., for transmembrane transport, motility, or synthesis of ATP by the ATP synthase. Results from a range of studies indicate that the membrane plays an important role in these processes, in addition to serving as a barrier. The membrane surface may also provide a proton link between the various membrane-embedded proteins, where the mere two-dimensional confinement of the reactants can also play a role. Enhancement of reaction rates between solute molecules and their target molecules on the surface and in the vicinity of biological membrane interfaces (e.g. ligand binding to membrane proteins) has been demonstrated in several studies and explained in terms of initial nonspecific binding of the solute molecules to the membrane followed by diffusion along the surface to their target molecules (1-8). Studies on some specific membrane-bound proton pumps, for example cytochrome c oxidase or bacteriorhodopsin (9-12), have revealed higher than diffusion-limited rates of proton upta...

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4

“…A section through a c 6 segment of the B. pseudofirmus OF4 c 13 ring with marked Cα positions of the alanines within the alkaliphilespecific 16 AxAxAxA 22 stretch shows this motif as a notable feature and the tight c-subunit packing (Fig. 1B).…”

Functional interactions between membrane-bound transporters and membranes

“…2, upon reconstitution of CytcO into the membrane, the rate constant increased from ∼830 s −1 to ∼5;900 s −1 (at pH 9.5), i.e., by a factor of 7, in agreement with an earlier study with the P. denitrificans CytcO (33). We per- formed the studies at high pH (9.5) because at this pH, the extent of the PCET displays a maximum and also the effect of the membrane on the proton-transfer rate increases with increasing pH (34). To test whether the acceleration of the PCET was due to a change in conductivity of the proton pathway or due to specific interactions with the membrane surface, we investigated the PCET in a number of mutant CytcOs in which residues within the pathway or at the surface near the pathway orifice were modified (summarized in Table 1).…”

Studying Ion Exchange in Solution and at Biological Membranes by FCS

“…The recent work by our group (Brändén et al 2006), (Öjemyr et al, 2009), (Persson et al, 2009), (Sandén et al, 2010) …”

“…). In analogy with this, similar charge screening effects have been analysed by FCS for protonation kinetics at biological membranes (Sandén et al, 2010) and for fluorescence quenching of fluorophores close to dielectric interfaces (Blom et al, 2010). However, since this charge screening affects the interaction between buffer molecules and fluorophores, no particular effect of ionic strength on k P is seen in the absence of a buffer, as illustrated in the inset of figure 2B, showing the relaxation rates measured at different concentrations of sodium chloride, with no buffer added.…”

“…Extending the range of pH over which the protonation kinetics of fluorescein-labelled lipids in liposomes were measured, two different proton exchange regimes could be identified (Sandén et al, 2010). At high pH (>8) the k +1 rate strongly increases with increasing proton concentrations, whereas at low pH (<7) the increase of the k +1 rate with increasing bulk [H + ]…”