Coupling to the proton gradient

• The "membrane voltmeters", - electrochromic response of the light-harvesting pigments

  The electrochromic respones of pigments in the light harvesting complexes were first identified as such by Horst Witt and Wolfgang Junge, working with chloroplasts in the late 1960s. They characterized a very rapid absorbance change centered around 515 nm that appeared to be a red shift in the spectrum of carotenoid and chlorophyll b, which was dependent on electron transfer through the two photosystems. The decay of this change was very slow, and unrelated to the decays of the electron transfer processes. The decay could be greatly accelarated by addition of ionophores with appropriate ionic partners to allow transfer of charge across the membrane. These characteristics identified the change as linked to the proton gradient, and Junge and Witt suggested that the mechanism was a well-known physical process, - the Stark effect, - more generally known as electrochromism. Electrochromism refers to the change in absorbance maximum of a colored species on application of an electric field. The theory is discussed in greater detail below. Deslome showed that under appropriate conditions, the rapid 515 nm change seen by Junge et al. showed an additional slow phase of increase, later characterized as reflecting turnover of the b₆f complex.

  A large absorbance change with similar characteristics had been identified earlier in the photosynthetic bacterium Rhodobacter sphaeroides by Jan Amesz, who had shown that the change could be modelled by a small red shift of the carotenoid absorbance bands. A similar change had been investigated in Rhodospirillum rubrum by Meg Baltscheffsky and Britton Chance. Taking advantage of the lower permeability of chromatophore membranes, and a familiarity with ionophoric mechanisms, Jackson and Crofts were able to demonstrate that the carotenoid electrochromic changes in Rb. sphaeroides were proportional to membrane potential. They used the ionophore valinomycin, which renders membranes specifically permeable to K⁺-ion, and thus allows membrane potentials of known magnitude to be generated by setting up gradients of K⁺ (as KCl) before addition of valinomycin.

  The electrochromic changes were easier to characterize in the photosynthetic bacteria because the carotenoid absorbance bands were well separated from those of the bacteriochlorophylls. Furthermore, mutant strains were developed that had fewer carotenoids (the G1C strain had Got 1 Carotenoid), which greatly simplified spectral analysis. Work from two competing groups identified the main characteristics of the absorbance change, and let to the recognition of some interesting paradoxes. Holmes and Crofts showed by careful measurment of the shift as a function of amplitude that the shift was very much smaller than expected from a simple electrochromic effect, and started at a point in the spectrum to the red of the bulk carotenoid absorbance. This was especially apparent on integration of the light minus dark difference spectrum (showing the shift), which showed that the spectrum of the shifted component was to the red. Amesz and de Grooth analyzed in great detail the kinetics of changes close to the pseudo-isobest of the difference spectrum, and showed that the change was indeed a shift. The results could be explained by suggesting that the absorbance change was a small shift of the carotenoid spectrum, but that the carotenoid population was not homogeneous. This inhomogeneity must reflect at least two populations, one of which (the active population) was red-shifted with respect to the other.

  Electrochromic Theory

  A second paradox came from analysis of the chemical nature of the species undergoing a shift, especially in Rb. sphaeroides G1C. In this strain, the carotenoid compement was 96% neurosporene, an apolar carotenoid. To understand the significance of this, it is necessary to explain a little electrochromic theory.

  The frequency shift associated with an electronic transition, Dn (in cm^-1), which is experienced by a molecule in an electric field, is given by:

  Dn = 1/hc ( -Dm · F - ½ Da · F² ) The terms Dm and Da denote, respectively, the difference between gound- and excited-state dipole moments and polarizabilities. Dm = m_e - m_g is the change in dipole moment on excitation; Da = a_e - a_g is the change in polarizability on excitation, F is the electric field, and F is the vector of the electric field in the direction of the dipole moment difference. The shift has two components, - a term linear with field strength (-Dm · F), which dominates for molecules with a permanent dipole, and a term quadratic with field strength (½ Da · F² ), which is the only term applicable for molecules with no permanent dipole.

  In Rb. sphaeroides GIC, the carotenoid molecule which showed an electrochromic shift was neurosporene, which is known to have no permanent dipole, and yet, as in the wild-type, experiments with valinomycin-induced K⁺ diffusion potentials showed that the shift was linear with field strength. This could be explained by suggesting that the carotenoids were experiencing a local field from the protein environment of the light-havesting complex, which polarized them, to give an induced dipole. An appropriate expression for the shift would include the polarizing local field, F_L, and the vector of the delocalized field in the direction of the induced dipole, F_D, which gives rise to the observed linear shift, as separate terms:

  Dn = 1/hc (- ½ Da · F_L · F_D)
  Solution of the crystallographic structures of light harvesting complexes from a similar bacteria has opened interesting possibilities for a deeper understanding of the electrochromic effect.

  The electrochromic changes in Rb. sphaeroides and the closely related Rb. capsulatus have been explored in detail. A fast phase associated with turnover of the photochemical reaction center, and immediate secondary donors and acceptors was resolved into two phases. Phase I was very rapid, and linked to the photochemistry; phase II was in the range 30-100 ms, and was associated with secondary electron transfer. A slower phase (phase III) was associated with turnover of the bc₁ complex, as shown by its sensitivity to specific inhibitors such as antimycin and myxothiazol. This later phase has been much studies in the context of the Q-cycle mechanism, since it reflects the electrogenic processes associated with turnover of the complex.

• Delayed fluorescence as a measure of energy storage in photosynthesis, and coupling to the proton gradient

  When plants that have been illuminated are returned to the dark, they emit a very faint light with the same spectrum as the fluorescence observed during illumination. While the latter (prompt fluorescence) has a life-time in the range 2-3 ns, the former (delayed fluorescence, or delayed light emission) may persists (with increasing loss of intensity) for minutes or hours. If illuminated materials are cooled, and then observed in the dark during warming, the light is emitted in bursts during the warming, - the so-called glow curves of thermoluminescence. These phenomena were discovered by Bill Arnold, Walter Bertsch, Lavorel and others in the 1960s, and recognized as related to the energy depth of the traps involved in the photochemical reactions. In the late 1960s and early 1970s, work from Darrell Fleischmann, Berger Mayne and Rod Clayton showed that delayed fluorescence was modulated by the "energized" state of the chloroplasts (the "high-energy" state linked to photophosphorylation). An explanation for these phenomena in terms of the chemiosmotic hypothesis came from the work of Wraight and Crofts, and from Barber and Kraan, from an explicit recognition of the role of the proton gradient, of the asymmetric organization of photosystem II across the coupling membrane (and hence the electrogenic nature of the photochemical reactions), and of the dependence of the secondary donor and acceptor reactions on the pH of the lumenal and stromal phases respectively, through their protolytic nature.

• Rapid H⁺ uptake and release in protolytic reactions.

  Many of the catalytic sites of photosynthetic electron transfer are involved in reactions in which the electron transfer is accompanied by release or uptake of a proton. These processes give rise to small changes in pH in the phase in which they occur. These changes can be measured with good accuracy by following the absorbance changes of pH indicator dyes, as long as the buffering capacity of the medium is kept low. By appropriate calibration using addition of acid or base of known concentration, the changes can be quantified, and related to the electron transfer changes with which they are linked by measurement of the latter using other biophysical approaches.

  Reactions that have been investigated using this approach are:

  • Release of protons accompanying the transitions of the S-states in oxygen evolution.
    In chloroplasts at neutral pH, or in isolated photosystem II (as long as the extrinsic peptides of the oxygen elvolving complex are retained), the pattern observed is 1, 0, 1, 2 H⁺ released on transitions S₀® S₁; S₁® S₂; S₂® S₃; and S₃® S₀ respectively. This pattern changes with pH, so that at low pH a pattern of 1, 1, 1, 1 is seen. A similar pattern is seen in stripped-down photosystem II preparations, especially if the extrinsic subunits are removed. This pattern of behavior has been variously interpreted, depending on the model for the mechanism of O₂ evolution preferred by the author (see Lecture 25). The pH dependence suggests that at least for the S₁® S₂ transition, protein side chains with pK values in the acidic range undergo dissociation, possible induced through a coulombic mechanism by charge changes within the Mn-complex.
    The kinetics of these changes have been studied in some detail, and it is found that for some transitions, the release of H⁺ is not kinetically matched with any one electron transfer step. For models in which all proton release is expected on oxidation of tyrosine Y_Z, a similar stoichiometry and kinetics matched to Y_Z oxidation would be expected on each transition. This is clearly not the case, - the observed kinetics are more similar to the S-state transition kinetics, but some phases are more rapid and likely reflect the faster events. This is difficult to reconcile with mechanism requiring a strict coupling.

  • The two-electron gate of photosystem II.
    In the neutral pH range, each flash generates an uptake of 1 H⁺. At extremes of the pH range 5-9, a binary pattern emerges, with less H⁺ uptake on the odd-numbered flashes. This behavior is consistent with a mechanism such as that discussed in a previous lecture, in which the semiquinone generated on the first (and odd-numbered flashes) is stabilized by uptake of a proton onto a neighboring protein side chain. This is reflected in the pH dependence of the equilibrium constant for sharing an electron between Q_A and Q_B, which could be interpeted as reflecting predominantly a single group. The binary pattern of H⁺ uptake as a function of pH matches quite well that expected from the pK values found from the pH dependence of the equilibrium constant. Below a pK of ~6, the group is already protonated, so no uptake is seen; above a pK of ~8, the group cannot be protonated, so no proton uptake is seen. The difference between these pK values reflects a change in pK induced on the group by the coulombic effect of the change in charge at the Q_B-site on formation of Q_B^.-. Recent mutagenesis studies suggest that the main group involved is His-252 of the D1 subunit.

  • The two-electron gate of bacterial reaction centers.
    This reaction is perhaps the most extensively studied example, and has generated an extensive literature. In general, the behavior is similar to that seen in photosystem II, but with complications arising from the fact that several protein side chains are involved. From mutagenesis studies, Glu-212 and Asp-213 of the L-subunit, close to the Q_B-binding domain, are the main players, but additional complications arise because of the pathway for proton access to the site through the H-subunit. The interested reader is referred to recent reviews:
    1. Alexo, E.G. and Gunner, M.R. (1999). Calculated protein and proton motions coupled to electron trans-fer: Electron transfer from QA- to QB in bacterial photosynthetic reaction centers. Biochemistry, 38, 8253-8270
    2. Graige, M.S., Paddock, M.L., Feher, G. and Okamura, M.Y. (1999). Observation of the protonated semi-quinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: Implications for the mechanism of electron and proton transfer in proteins. Biochemistry 38, 11465-11473.

  • The Q_i-site of the bc₁ complex.
    Proton uptake on reduction of ubiquinone at the Q_i-site can be assayed from the antimycin sensitive phase of the rapid H⁺ uptake following flash excitation of chromatophores. Extensive work has been reported on the dependence of this phase on the initial redox state of the system, and has shown the uptake of protons at the site expected from the Q-cycle mechanism, with kinetics similar to that of the rate limiting step of quinol oxidation.

  • The Q_o-site of the bc₁ complex. Because the Q_o-site is exposed to the interior of the chromatophore, measurement of the H⁺ release expected from the Q-cycle mechanism is more difficult, and has been less studied. Two approaches have been used:
    a) measurement of absorbance changes of neutral red, which, in the presence of external non-permeant buffers, monitors mainly the pH change inside chromatophores; and
    b) measurement in cells, or spheroplasts derived by lysozyme treatment of cells to remove the outer membrane.
    In general, the results showed the myxothiazol-sensitive H⁺ release expected, and the kinetics were similar to the re-reduction of cyt c₂. However, in cells a much slower rate was seen for the H⁺ release than for the electron transfer reaction, and the "results (were) discussed in the context of the hypothesis that H⁺ release after flash excitation of intact cells is restricted by immobile buffering groups in the periplasm and outer membrane."

• The electrogenic reactions of photosynthesis explored using the electrochromic changes.

• Flux through ATP synthase explored using electrochromic changes.

• Fluorescence "lowering", - later rediscovered as nonphotochemical quenching, - and the protection of plants against excess light.