New and Notable
Proton Dynamics at the Membrane Surface Robert B. Gennis1,* 1
Department of Biochemistry, University of Illinois, Urbana, Illinois
Proton transfer over large distances is critical to biology, particularly so for cellular bioenergetics. Transmembrane proton pumps such as cytochrome oxidase generate a proton electrochemical gradient or proton motive force (D~ mH þ or PMF) across a membrane (e.g., mitochondrial inner membrane or bacterial cytoplasmic membrane) while other transmembrane proteins, such as the F1F0-ATP synthase, the flagellar motor, or secondary transporters, utilize the PMF to drive otherwise unfavorable reactions (e.g., ATP synthesis). A steady-state proton current is established from the proton pumps to the ATP synthases (as an example) on the electrically positive side of the membrane and then back again on the negative side of the membrane. But what are the dominant pathways for proton flux between the generators and utilizers of the PMF? Essentially, the question is whether protons diffuse along the membrane surface between proton sources and sinks, or if protons generated at the surface rapidly equilibrate with the bulk solution. Equilibration of surface-confined protons with the bulk solution involves a loss of free energy. If protons remain confined to the membrane surface, the free energy available to drive ATP synthesis will be larger.
Submitted March 29, 2016, and accepted for publication April 1, 2016. *Correspondence: [email protected] Editor: Claudia Steinem.
Over the past decade, experimental, theoretical, and computational approaches have progressed in the description of proton dynamics at the membrane surface to the point where they seem to be approaching a consensus view (1,2). The article by Xu et al. (3) in this issue of the Biophysical Journal makes important contributions to this topic. This article is the latest in a series (4–6) from the Brzezinski and Widengren groups in which they use fluorescence correlation spectroscopy (FCS) to analyze the fluctuations in fluorescence intensity of a pH-sensitive probe (fluorescein), yielding the on-rate and off-rate for protonation of the probe in different locations. Previously, the protonation kinetics of the fluorescein probe was examined for the probe in water, attached to a lipid headgroup within a phospholipid vesicle and attached to cytochrome oxidase embedded in a vesicle bilayer. In this work, the protonation kinetics of the lipid-attached and protein-attached fluorescein probes was examined in phospholipid nanodisks of different sizes. In all cases, the off-rate constant is essentially the same (koff ¼ 5 104 s1), but the on-rate constant changes over a wide range. In unbuffered water, the rate constant for the protonation of fluorescein is at the diffusion limit, kon ¼ 4 1010 M1 s1. When anchored to the surface of a phospholipid liposome, attached either to a lipid or membrane-protein, the rate constant in-
creases to ~kon ¼ 7.5 1012 M1 s1 for pH > 8, reflecting a local proton concentration at the membrane surface that is 100-fold higher than in the bulk. Under these conditions, the phospholipid surface acts as a ‘‘proton-collecting antenna’’, facilitating rapid protonation of groups at the membrane surface. In this work, it is shown that the rate-enhancement requires a minimal lipid surface area (the proton antenna) in the range of 60 nm2. When the lipid surface area is significantly less than this value, the protonation rate constant is only slightly larger than that of fluorescein in water, and the ‘‘proton-collecting antenna’’ effect vanishes. Larger surface areas do not further enhance the protonation rate. In addition, the influence of external mobile buffers is also investigated. Impressively, all the data, including both the dependence on bilayer area and buffer effects, can be fit using Monte Carlo simulations based on a relatively simple kinetic model. The conceptual framework contains important elements from recent theoretical considerations (2,7,8) and explains why the diffusion coefficient for protons along the surface varies over a range of 100-fold, from 5 105 cm2 s1 to 2 107 cm2 s1, depending on the method used to measure it. Although FCS measures protonation exchange rates under equilibrium conditions, the results are relevant to understanding the steady-state proton currents that are of biological significance. Much remains vague
http://dx.doi.org/10.1016/j.bpj.2016.04.001 Ó 2016 Biophysical Society.
Biophysical Journal 110, 1909–1911, May 10, 2016 1909
Gennis
and unclear, but a set of (over)simplified elements are given below. 1) Phospholipid bilayers have a narrow region (deep interface zone; Fig. 1) where the headgroup phosphates and carbonyls stabilize excess hydrated protons relative to the bulk solution by ~4 kcal/mol (7). The pH in this interface region is much more acidic than bulk solution (5,7).This does not require a net negative charge and is observed for bilayers composed of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylglycerol phospholipids. The protons are confined in water clusters within this deep interface zone and diffuse along with the lipid molecules as well as by hopping between clusters. As determined by FCS, the diffusion coefficient of these protons in the deep interface zone (DS ¼ 2 107 cm2 s1) is only 2–3-fold faster than that of phospholipids within the bilayer. 2) A protein residue or dye at the membrane surface can be protonated by 1) protons within the interface zone, or 2) protons within a layer of the bulk solution adjacent
to the surface, or 3) protonated buffer molecules within the adjacent bulk zone. 3) Protons in the deep interface zone can exchange with those in the adjacent bulk solution either by thermal fluctuations or, if present, by collision with mobile buffer molecules. 4) A proton pump generates a flux of excess protons at the membrane surface. We can consider two conditions: 1) Equilibration with the bulk solution is slow relative to the proton exchange rate with the surface. The limiting case is in the absence of buffers and this condition will also apply for buffer concentrations ~10 mM, the equilibration with the bulk is greatly accelerated. Protons generated at a source are rapidly grabbed at the membrane surface and dissipated within the bulk via buffer molecules. As a result, all surface effects vanish. Why is this important in biology? Mitchell’s chemiosmotic theory (12) assumes that the protons on either side of the membrane rapidly equilibrate with the bulk solution so that the free energy of transferring a proton from one side to the other is accurately measured by the difference in the electrochemical potential of protons in the bulk solutions on either side of the membrane ðD~ mHþ Þ, as follows: PMF zDj 60DpH at 298 K
FIGURE 1 Schematic phospholipid bilayer showing the regions referred to in the text. The deep interface zone is a thin layer at the level of the phosphate groups where the local proton concentration may be 100-fold greater than in the bulk solution. Excess protons generated by a pump will exchange with the adjacent bulk zone and then diffuse away from the membrane surface. Key parameters to understand proton dynamics are the time required to reach equilibration, and the characteristic time of proton exchange. To see this figure in color, go online.
1910 Biophysical Journal 110, 1909–1911, May 10, 2016
ðin mV unitsÞ:
(1)
The assumption of rapid protonic equilibration has been challenged from the start with variations of the
New and Notable
concept of localized proton transfer between proton sources and sinks without the necessity to equilibrate with the bulk solution, most notably by Williams (13,14). Experimentally, attempts over the years to demonstrate the biological significance of localized proton transfer in bioenergetics have largely failed or been ambiguous, with the prominent exception of studies on alkaliphilic bacteria that use the PMF to drive ATP synthesis when the external pH is 10.5 or even higher (15). For these alkaliphiles, the PMF is far too low to provide the required free energy to synthesize ATP. Yet, it is clear that proton flux does drive ATP synthesis. Thermodynamics excludes the possibility of equilibration of the pumped protons with the bulk solution (16). This work suggests a way out of this puzzle. Perhaps these organisms have evolved to avoid the dissipation of the free energy of their proton pumps by confining the pumped protons to the membrane surface (which includes the adjacent bulk layer), effectively lowering the local pH sensed by the ATP synthase on the positive side of the membrane so that the DpH term (between the two sides of the membrane) in Eq. 1 becomes favorable for ATP synthesis. This increase in the proton motive force would depend on the rate of proton exchange between the bulk and interface zone as well as the distance separating proton pumps and ATP synthase molecules. A change of 2 units of pH would be sufficient.
Interestingly, a deviation from the chemiosmotic principle in the opposite direction was recently demonstrated for the mitochondrion (17). The proton pumps and ATP synthase molecules are located in different regions of the christae separated by ~0.5 mm. Steady-state proton diffusion between the proton sources and sinks appears to be rapid, assumed to be through the bulk solution, but is still ratelimiting for the process. As a result, a lateral pH-gradient is established between the two regions such that the pH sensed by the ATP synthase molecules is higher by 0.3 pH units. This effectively reduces the proton motive force under some physiological conditions, though not enough to be significant. REFERENCES 1. Medvedev, E. S., and A. A. Stuchebrukhov. 2011. Proton diffusion along biological membranes. J. Phys. Condens. Matter. 23:1–15. 2. Wolf, M. G., H. Grubmu¨ller, and G. Groenhof. 2014. Anomalous surface diffusion of protons on lipid membranes. Biophys. J. 107:76–87. ¨ jemyr, ., J. Widengren. 3. Xu, L., L. N. O 2016. Protonation dynamics on lipid nanodisks—influence of the membrane surface area and external buffers. Biophys. J. 110:1993–2003. 4. Sande´n, T., L. Salomonsson, ., J. Widengren. 2010. Surface-coupled proton exchange of a membrane-bound proton acceptor. Proc. Natl. Acad. Sci. USA. 107:4129–4134. 5. Ojemyr, L., T. Sande´n, ., P. Brzezinski. 2009. Lateral proton transfer between the membrane and a membrane protein. Biochemistry. 48:2173–2179.
6. Bra¨nde´n, M., T. Sande´n, ., J. Widengren. 2006. Localized proton microcircuits at the biological membrane-water interface. Proc. Natl. Acad. Sci. USA. 103:19766–19770. 7. Yamashita, T., and G. A. Voth. 2010. Properties of hydrated excess protons near phospholipid bilayers. J. Phys. Chem. B. 114: 592–603. 8. Medvedev, E. S., and A. A. Stuchebrukhov. 2013. Mechanism of long-range proton translocation along biological membranes. FEBS Lett. 587:345–349. 9. Serowy, S., S. M. Saparov, ., P. Pohl. 2003. Structural proton diffusion along lipid bilayers. Biophys. J. 84:1031–1037. 10. Springer, A., V. Hagen, ., P. Pohl. 2011. Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface. Proc. Natl. Acad. Sci. USA. 108: 14461–14466. 11. Mulkidjanian, A. Y., J. Heberle, and D. A. Cherepanov. 2006. Protons at interfaces: implications for biological energy conversion. Biochim. Biophys. Acta. 1757: 913–930. 12. Mitchell, P. 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 191:144–148. 13. Williams, R. J. 1978. The history and the hypotheses concerning ATP-formation by energised protons. FEBS Lett. 85:9–19. 14. Williams, R. J. 1988. Proton circuits in biological energy interconversions. Annu. Rev. Biophys. Biophys. Chem. 17:71–97. 15. Preiss, L., J. D. Langer, ., T. Meier. 2014. The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle. Mol. Microbiol. 92:973–984. 16. Krulwich, T. A. 1995. Alkaliphiles: ‘basic’ molecular problems of pH tolerance and bioenergetics. Mol. Microbiol. 15:403–410. 17. Rieger, B., W. Junge, and K. B. Busch. 2014. Lateral pH gradient between OXPHOS complex IV and F0F1 ATP-synthase in folded mitochondrial membranes. Nat. Commun. 5:3103.
Biophysical Journal 110, 1909–1911, May 10, 2016 1911
Proton Dynamics at the Membrane Surface Robert B. Gennis1,* 1
Department of Biochemistry, University of Illinois, Urbana, Illinois
Proton transfer over large distances is critical to biology, particularly so for cellular bioenergetics. Transmembrane proton pumps such as cytochrome oxidase generate a proton electrochemical gradient or proton motive force (D~ mH þ or PMF) across a membrane (e.g., mitochondrial inner membrane or bacterial cytoplasmic membrane) while other transmembrane proteins, such as the F1F0-ATP synthase, the flagellar motor, or secondary transporters, utilize the PMF to drive otherwise unfavorable reactions (e.g., ATP synthesis). A steady-state proton current is established from the proton pumps to the ATP synthases (as an example) on the electrically positive side of the membrane and then back again on the negative side of the membrane. But what are the dominant pathways for proton flux between the generators and utilizers of the PMF? Essentially, the question is whether protons diffuse along the membrane surface between proton sources and sinks, or if protons generated at the surface rapidly equilibrate with the bulk solution. Equilibration of surface-confined protons with the bulk solution involves a loss of free energy. If protons remain confined to the membrane surface, the free energy available to drive ATP synthesis will be larger.
Submitted March 29, 2016, and accepted for publication April 1, 2016. *Correspondence: [email protected] Editor: Claudia Steinem.
Over the past decade, experimental, theoretical, and computational approaches have progressed in the description of proton dynamics at the membrane surface to the point where they seem to be approaching a consensus view (1,2). The article by Xu et al. (3) in this issue of the Biophysical Journal makes important contributions to this topic. This article is the latest in a series (4–6) from the Brzezinski and Widengren groups in which they use fluorescence correlation spectroscopy (FCS) to analyze the fluctuations in fluorescence intensity of a pH-sensitive probe (fluorescein), yielding the on-rate and off-rate for protonation of the probe in different locations. Previously, the protonation kinetics of the fluorescein probe was examined for the probe in water, attached to a lipid headgroup within a phospholipid vesicle and attached to cytochrome oxidase embedded in a vesicle bilayer. In this work, the protonation kinetics of the lipid-attached and protein-attached fluorescein probes was examined in phospholipid nanodisks of different sizes. In all cases, the off-rate constant is essentially the same (koff ¼ 5 104 s1), but the on-rate constant changes over a wide range. In unbuffered water, the rate constant for the protonation of fluorescein is at the diffusion limit, kon ¼ 4 1010 M1 s1. When anchored to the surface of a phospholipid liposome, attached either to a lipid or membrane-protein, the rate constant in-
creases to ~kon ¼ 7.5 1012 M1 s1 for pH > 8, reflecting a local proton concentration at the membrane surface that is 100-fold higher than in the bulk. Under these conditions, the phospholipid surface acts as a ‘‘proton-collecting antenna’’, facilitating rapid protonation of groups at the membrane surface. In this work, it is shown that the rate-enhancement requires a minimal lipid surface area (the proton antenna) in the range of 60 nm2. When the lipid surface area is significantly less than this value, the protonation rate constant is only slightly larger than that of fluorescein in water, and the ‘‘proton-collecting antenna’’ effect vanishes. Larger surface areas do not further enhance the protonation rate. In addition, the influence of external mobile buffers is also investigated. Impressively, all the data, including both the dependence on bilayer area and buffer effects, can be fit using Monte Carlo simulations based on a relatively simple kinetic model. The conceptual framework contains important elements from recent theoretical considerations (2,7,8) and explains why the diffusion coefficient for protons along the surface varies over a range of 100-fold, from 5 105 cm2 s1 to 2 107 cm2 s1, depending on the method used to measure it. Although FCS measures protonation exchange rates under equilibrium conditions, the results are relevant to understanding the steady-state proton currents that are of biological significance. Much remains vague
http://dx.doi.org/10.1016/j.bpj.2016.04.001 Ó 2016 Biophysical Society.
Biophysical Journal 110, 1909–1911, May 10, 2016 1909
Gennis
and unclear, but a set of (over)simplified elements are given below. 1) Phospholipid bilayers have a narrow region (deep interface zone; Fig. 1) where the headgroup phosphates and carbonyls stabilize excess hydrated protons relative to the bulk solution by ~4 kcal/mol (7). The pH in this interface region is much more acidic than bulk solution (5,7).This does not require a net negative charge and is observed for bilayers composed of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylglycerol phospholipids. The protons are confined in water clusters within this deep interface zone and diffuse along with the lipid molecules as well as by hopping between clusters. As determined by FCS, the diffusion coefficient of these protons in the deep interface zone (DS ¼ 2 107 cm2 s1) is only 2–3-fold faster than that of phospholipids within the bilayer. 2) A protein residue or dye at the membrane surface can be protonated by 1) protons within the interface zone, or 2) protons within a layer of the bulk solution adjacent
to the surface, or 3) protonated buffer molecules within the adjacent bulk zone. 3) Protons in the deep interface zone can exchange with those in the adjacent bulk solution either by thermal fluctuations or, if present, by collision with mobile buffer molecules. 4) A proton pump generates a flux of excess protons at the membrane surface. We can consider two conditions: 1) Equilibration with the bulk solution is slow relative to the proton exchange rate with the surface. The limiting case is in the absence of buffers and this condition will also apply for buffer concentrations ~10 mM, the equilibration with the bulk is greatly accelerated. Protons generated at a source are rapidly grabbed at the membrane surface and dissipated within the bulk via buffer molecules. As a result, all surface effects vanish. Why is this important in biology? Mitchell’s chemiosmotic theory (12) assumes that the protons on either side of the membrane rapidly equilibrate with the bulk solution so that the free energy of transferring a proton from one side to the other is accurately measured by the difference in the electrochemical potential of protons in the bulk solutions on either side of the membrane ðD~ mHþ Þ, as follows: PMF zDj 60DpH at 298 K
FIGURE 1 Schematic phospholipid bilayer showing the regions referred to in the text. The deep interface zone is a thin layer at the level of the phosphate groups where the local proton concentration may be 100-fold greater than in the bulk solution. Excess protons generated by a pump will exchange with the adjacent bulk zone and then diffuse away from the membrane surface. Key parameters to understand proton dynamics are the time required to reach equilibration, and the characteristic time of proton exchange. To see this figure in color, go online.
1910 Biophysical Journal 110, 1909–1911, May 10, 2016
ðin mV unitsÞ:
(1)
The assumption of rapid protonic equilibration has been challenged from the start with variations of the
New and Notable
concept of localized proton transfer between proton sources and sinks without the necessity to equilibrate with the bulk solution, most notably by Williams (13,14). Experimentally, attempts over the years to demonstrate the biological significance of localized proton transfer in bioenergetics have largely failed or been ambiguous, with the prominent exception of studies on alkaliphilic bacteria that use the PMF to drive ATP synthesis when the external pH is 10.5 or even higher (15). For these alkaliphiles, the PMF is far too low to provide the required free energy to synthesize ATP. Yet, it is clear that proton flux does drive ATP synthesis. Thermodynamics excludes the possibility of equilibration of the pumped protons with the bulk solution (16). This work suggests a way out of this puzzle. Perhaps these organisms have evolved to avoid the dissipation of the free energy of their proton pumps by confining the pumped protons to the membrane surface (which includes the adjacent bulk layer), effectively lowering the local pH sensed by the ATP synthase on the positive side of the membrane so that the DpH term (between the two sides of the membrane) in Eq. 1 becomes favorable for ATP synthesis. This increase in the proton motive force would depend on the rate of proton exchange between the bulk and interface zone as well as the distance separating proton pumps and ATP synthase molecules. A change of 2 units of pH would be sufficient.
Interestingly, a deviation from the chemiosmotic principle in the opposite direction was recently demonstrated for the mitochondrion (17). The proton pumps and ATP synthase molecules are located in different regions of the christae separated by ~0.5 mm. Steady-state proton diffusion between the proton sources and sinks appears to be rapid, assumed to be through the bulk solution, but is still ratelimiting for the process. As a result, a lateral pH-gradient is established between the two regions such that the pH sensed by the ATP synthase molecules is higher by 0.3 pH units. This effectively reduces the proton motive force under some physiological conditions, though not enough to be significant. REFERENCES 1. Medvedev, E. S., and A. A. Stuchebrukhov. 2011. Proton diffusion along biological membranes. J. Phys. Condens. Matter. 23:1–15. 2. Wolf, M. G., H. Grubmu¨ller, and G. Groenhof. 2014. Anomalous surface diffusion of protons on lipid membranes. Biophys. J. 107:76–87. ¨ jemyr, ., J. Widengren. 3. Xu, L., L. N. O 2016. Protonation dynamics on lipid nanodisks—influence of the membrane surface area and external buffers. Biophys. J. 110:1993–2003. 4. Sande´n, T., L. Salomonsson, ., J. Widengren. 2010. Surface-coupled proton exchange of a membrane-bound proton acceptor. Proc. Natl. Acad. Sci. USA. 107:4129–4134. 5. Ojemyr, L., T. Sande´n, ., P. Brzezinski. 2009. Lateral proton transfer between the membrane and a membrane protein. Biochemistry. 48:2173–2179.
6. Bra¨nde´n, M., T. Sande´n, ., J. Widengren. 2006. Localized proton microcircuits at the biological membrane-water interface. Proc. Natl. Acad. Sci. USA. 103:19766–19770. 7. Yamashita, T., and G. A. Voth. 2010. Properties of hydrated excess protons near phospholipid bilayers. J. Phys. Chem. B. 114: 592–603. 8. Medvedev, E. S., and A. A. Stuchebrukhov. 2013. Mechanism of long-range proton translocation along biological membranes. FEBS Lett. 587:345–349. 9. Serowy, S., S. M. Saparov, ., P. Pohl. 2003. Structural proton diffusion along lipid bilayers. Biophys. J. 84:1031–1037. 10. Springer, A., V. Hagen, ., P. Pohl. 2011. Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface. Proc. Natl. Acad. Sci. USA. 108: 14461–14466. 11. Mulkidjanian, A. Y., J. Heberle, and D. A. Cherepanov. 2006. Protons at interfaces: implications for biological energy conversion. Biochim. Biophys. Acta. 1757: 913–930. 12. Mitchell, P. 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 191:144–148. 13. Williams, R. J. 1978. The history and the hypotheses concerning ATP-formation by energised protons. FEBS Lett. 85:9–19. 14. Williams, R. J. 1988. Proton circuits in biological energy interconversions. Annu. Rev. Biophys. Biophys. Chem. 17:71–97. 15. Preiss, L., J. D. Langer, ., T. Meier. 2014. The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle. Mol. Microbiol. 92:973–984. 16. Krulwich, T. A. 1995. Alkaliphiles: ‘basic’ molecular problems of pH tolerance and bioenergetics. Mol. Microbiol. 15:403–410. 17. Rieger, B., W. Junge, and K. B. Busch. 2014. Lateral pH gradient between OXPHOS complex IV and F0F1 ATP-synthase in folded mitochondrial membranes. Nat. Commun. 5:3103.
Biophysical Journal 110, 1909–1911, May 10, 2016 1911
