Eur Biophys J DOI 10.1007/s00249-015-1013-1
ORIGINAL PAPER
Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers Fulvio Ciriaco · Rocco Roberto Tangorra · Alessandra Antonucci · Livia Giotta · Angela Agostiano · Massimo Trotta · Francesco Milano
Received: 2 December 2014 / Revised: 4 February 2015 / Accepted: 5 February 2015 © European Biophysical Societies’ Association 2015
Abstract Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant KQ at the QB site. The proposed approach provides for the first time the evaluation of KQ without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure. Keywords Reaction · Centers · Semiquinone oscillations · Ligand equilibrium constant · Quinone binding
Electronic supplementary material The online version of this article (doi:10.1007/s00249-015-1013-1) contains supplementary material, which is available to authorized users. F. Ciriaco · R. R. Tangorra · A. Antonucci · A. Agostiano Department of Chemistry, University of Bari, 70126 Bari, Italy
Introduction The photosynthetic reaction center (RC) from the bacterium Rhodobacter (R.) sphaeroides is the pivotal protein in the photosynthetic process, catalyzing the ubiquinone reduction to ubiquinol by drawing electrons from reduced cytochrome c2, thereby converting radiant energy into chemical energy. Progress in understanding its functioning is of paramount importance in several fields such as biophysics, optoelectronics, biosensing and so on. From a structural point of view, RC is a membranespanning protein composed of three subunits named L, M, and H. Nine cofactors are found in the protein scaffold: two ubiquinone-10 (UQ10) molecules (located in two binding pockets called QA and QB), an iron ion, two bacteriopheophytins (BΦ) and four bacteriochlorophylls (Bchl), two of which form a functional dimer (D). The purified RC is surrounded by a toroid of detergent molecules (Roth et al. 1989) preventing its precipitation in water. Upon photon absorption, D is promoted to its singlet excited state, and one electron is shuttled to the electron acceptor QA. A fast electron exchange reaction occurs between the two quinones with equilibrium constant KE. The two ubiquinones are thus better described as a single entity called the quinone acceptor complex and denoted as (QAQB). In the absence of external reductants, the following charge recombination (CR) reaction occurs:
L. Giotta Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, 73100 Lecce, Italy
D+ (QA QB )− → D(QA QB ).
A. Agostiano · M. Trotta · F. Milano (*) Italian National Research Council, Institute for Physical and Chemical Processes (CNR-IPCF), 70125 Bari, Italy e-mail: [email protected]
D+ Q− A → DQA
(1)
When the QB site is empty or occupied by an inhibitor, the electron recombines from QA: (2)
Reaction (2) occurs via electron tunneling, and its kinetic constant, almost independent on the temperature, is
13
Eur Biophys J
kAD = 10 s−1. Reaction (1) is more complex as it occurs via “thermal repopulation” of QA with kinetic constant kS approximately related to KE and kAD by the following equation (Shinkarev and Wraight 1993):
kS = kAD /(1 + KE )
(3)
measurement of kS is therefore a way to infer the KE value. Progressive repopulation of the QB site, adding increasing amounts of exogenous quinone to QB-depleted RC, allows determining the quinone binding constant KQ. This is usually obtained by recording the CR traces at each quinone concentration and correlating the fraction of active QB sites to the parameters of the relevant biexponential function fitting the data, i.e., the fraction of the slow phase, its time constant or a combination of them. Details, potentialities and limitations of this approach are given in (Mavelli et al. 2014). Two main differences characterize the quinones QA and QB: the former, firmly bound to the protein, is a cofactor able to accept only one electron, while the latter, loosely bound, acts as a substrate accepting two electrons. In the presence of an exogenous electron donor to D+ (physiologically represented by cytochrome c2), a second photon absorption triggers the transfer of a second electron to the quinone complex, so that the final acceptor quinone QB, upon double reduction and protonation, is released and substituted by a new quinone molecule from an exogenous pool (Feher et al. 1989). This photocycle can be reconstituted in solution and driven as long as the exogenous pools of either the external electron donor or acceptor run out (Milano et al. 2007). In these conditions, a sequence of flashes generates the so-called semiquinone oscillations because of the appearance of semiquinone at odd flashes and its disappearance at even flashes in the form of ubiquinol. The resulting flash-induced rise and drop of absorb− ance at 450 nm, where the reduced species (DQ− A, DQA QB − and DQA QB) absorb with a similar extinction coefficient (Kleinfeld et al. 1985), are typical spectroscopic behaviors of ubiquinone-based RCs and represent a valuable source of information on the kinetics and thermodynamics of ubiquinone reactions within these proteins (Vermeglio 1977; Wraight 1977). The first quantitative interpretation of absorbance changes was conducted on data obtained in the presence of a suitable electron donor, ensuring the complete D+ re-reduction before any charge recombination, and with excess ubiquinone concentration (Kleinfeld et al. 1984). Oscillations were damped since, at each step, a fraction 1/(1 + KE) of RCs is found in the inactive state DQ− A QB. Under these conditions, mutual linear relationships exist between the concentrations of different RC redox states at consecutive flashes, and the relevant system of linear equations can be solved analytically (Kleinfeld et al. 1984). In
13
order to properly fit the experimental data, the only adjustable parameters are the amplitude of semiquinone absorption after the first flash (ΔA1) and the interquinone electron transfer equilibrium constant KE according to the equation: m KE 1 + 2KE m �A1 1 − (−1) �Am = (4) 1 + KE 1 + KE where m is the flash number. This approach presents some limitations: • It is not suitable for improving the accuracy of the KE determination exploiting further information such as the different molar extinction coefficients of charged and uncharged species that are either available in the literature or can be inferred from ad hoc experiments. The above-mentioned procedure completely disregards this kind of information, possibly affecting the obtained KE value. • The flash sequence technique can also be exploited to obtain other parameters such as the quinone binding constant. However, to this end, one has to work with quinone concentrations leading to partial occupation of the available QB sites (subsaturating condition). • Various effects inherent to the experimental setup (excitation from the measuring light, quinole reoxidation, subsaturating flash light) appear in the recorded traces, which can strongly deviate from the ideal law described above. Disregarding these effects also can affect the meaning of the fitting parameters. In recent years, more advanced mathematical models for describing the oscillations also in the presence of inhibitors have been proposed (Halmschlager et al. 2002). In the present article, a more general kinetic model proposed by Shinkarev (1998) is implemented in a C-code used for numerical simulations of experimental data collected under sub-saturating ubiquinone concentrations. This condition induces a nonlinear oscillation pattern and enables evaluating the ubiquinone binding constant KQ, a parameter inaccessible working with saturated QB sites. Interestingly, unlike in the standard above-mentioned titration procedures, the use of the oscillation pattern does not impose depleting the QB site in the case of the native ubiquinone-10. In fact, 70–80 % of the QB sites are typically occupied in pristine RCs, which means that they are close to saturation in the CR essay. Conversely, oscillations lead naturally to a depletion of the QB sites and the relevant pattern changes even starting from completely filled QB sites. The advantage of the oscillation method resides hence in the possibility of avoiding harsh treatments of the protein, which may scramble the QB pocket. The binding constant KQ for UQ10 obtained in this article is hence the first, to our knowledge, obtained in a pristine QB site.
Eur Biophys J
Materials Chemicals All chemicals used in this work were purchased from Sigma-Aldrich and used without any further purification. Ubiquinone 0 (UQ0, 2,3-dimethoxy-5-methyl-1, 4-p-benzoubiquinone) stock solution was 40 mM in water. Ubiquinone 10 (UQ10) stock solution was 600 μM in 0.3 % Triton X-100 (TX). Ferrocene–methanol (FcnMeOH) stock solution was 10 mM in ethanol. Ferrocyanide (FeCy) stock solution was 100 mM in water. The pH-indicator absorption dye o-cresol red stock solution was 1 mM ethanol. All experiments were performed with freshly prepared solutions. Reaction centers RCs were isolated from blue-green strain R26 of the photosynthetic bacterium Rhodobacter (R.) sphaeroides following an established procedure using lauryldimethylamineN-oxide (LDAO) for extraction and ammonium sulfate precipitation and diethylaminoethyl (DEAE) chromatography for purification (Milano et al. 2009). The final ratio of absorbances at 280 and 802 nm was lower than 1.3 (optimal value for pure RC is 1.28), and the RC concentration was determined from the steady-state absorption at 802 nm using 288 mM−1 cm−1 as extinction coefficient. LDAO was exchanged with TX by overnight dialysis against 10 mM Tris buffer (pH 8.0), TX 0.03 % at 4 °C. The native UQ10 in the QB site was extracted with 1 % LDAO and 2 mM orthophenanthroline (Okamura et al. 1975). Steady‑state and transient optical spectroscopy The steady-state optical spectra were recorded by a Cary 5000 (Agilent) UV-visible-NIR spectrophotometer. Flashinduced absorbance changes were recorded using a kinetic spectrometer of local design described elsewhere (Milano et al. 2003).
Scheme 1 A general model of processes activated when reaction centers are subjected to light flashes in the presence of a fast electron donor. The waved arrows indicate processes happening upon flash excitation, double arrows denote fast chemical equilibria, and single arrows denote slower processes observable during the interval between two flashes
buffered sample (Maróti and Wraight 1988). The relevant data are presented in the supplementary material. Mathematical methods and fitting procedures Scheme 1 has been implemented in a C-code from the flow chart in figure S1 and used for generating simulated data sets and for fitting the relevant experimental data. The C-code, provided in a separate file, is described in the supplementary material along with additional details.
Results and discussion Modeling the oscillation pattern
Proton release measurements The pH indicator dye o-cresol red (7.5
ORIGINAL PAPER
Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers Fulvio Ciriaco · Rocco Roberto Tangorra · Alessandra Antonucci · Livia Giotta · Angela Agostiano · Massimo Trotta · Francesco Milano
Received: 2 December 2014 / Revised: 4 February 2015 / Accepted: 5 February 2015 © European Biophysical Societies’ Association 2015
Abstract Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant KQ at the QB site. The proposed approach provides for the first time the evaluation of KQ without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure. Keywords Reaction · Centers · Semiquinone oscillations · Ligand equilibrium constant · Quinone binding
Electronic supplementary material The online version of this article (doi:10.1007/s00249-015-1013-1) contains supplementary material, which is available to authorized users. F. Ciriaco · R. R. Tangorra · A. Antonucci · A. Agostiano Department of Chemistry, University of Bari, 70126 Bari, Italy
Introduction The photosynthetic reaction center (RC) from the bacterium Rhodobacter (R.) sphaeroides is the pivotal protein in the photosynthetic process, catalyzing the ubiquinone reduction to ubiquinol by drawing electrons from reduced cytochrome c2, thereby converting radiant energy into chemical energy. Progress in understanding its functioning is of paramount importance in several fields such as biophysics, optoelectronics, biosensing and so on. From a structural point of view, RC is a membranespanning protein composed of three subunits named L, M, and H. Nine cofactors are found in the protein scaffold: two ubiquinone-10 (UQ10) molecules (located in two binding pockets called QA and QB), an iron ion, two bacteriopheophytins (BΦ) and four bacteriochlorophylls (Bchl), two of which form a functional dimer (D). The purified RC is surrounded by a toroid of detergent molecules (Roth et al. 1989) preventing its precipitation in water. Upon photon absorption, D is promoted to its singlet excited state, and one electron is shuttled to the electron acceptor QA. A fast electron exchange reaction occurs between the two quinones with equilibrium constant KE. The two ubiquinones are thus better described as a single entity called the quinone acceptor complex and denoted as (QAQB). In the absence of external reductants, the following charge recombination (CR) reaction occurs:
L. Giotta Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, 73100 Lecce, Italy
D+ (QA QB )− → D(QA QB ).
A. Agostiano · M. Trotta · F. Milano (*) Italian National Research Council, Institute for Physical and Chemical Processes (CNR-IPCF), 70125 Bari, Italy e-mail: [email protected]
D+ Q− A → DQA
(1)
When the QB site is empty or occupied by an inhibitor, the electron recombines from QA: (2)
Reaction (2) occurs via electron tunneling, and its kinetic constant, almost independent on the temperature, is
13
Eur Biophys J
kAD = 10 s−1. Reaction (1) is more complex as it occurs via “thermal repopulation” of QA with kinetic constant kS approximately related to KE and kAD by the following equation (Shinkarev and Wraight 1993):
kS = kAD /(1 + KE )
(3)
measurement of kS is therefore a way to infer the KE value. Progressive repopulation of the QB site, adding increasing amounts of exogenous quinone to QB-depleted RC, allows determining the quinone binding constant KQ. This is usually obtained by recording the CR traces at each quinone concentration and correlating the fraction of active QB sites to the parameters of the relevant biexponential function fitting the data, i.e., the fraction of the slow phase, its time constant or a combination of them. Details, potentialities and limitations of this approach are given in (Mavelli et al. 2014). Two main differences characterize the quinones QA and QB: the former, firmly bound to the protein, is a cofactor able to accept only one electron, while the latter, loosely bound, acts as a substrate accepting two electrons. In the presence of an exogenous electron donor to D+ (physiologically represented by cytochrome c2), a second photon absorption triggers the transfer of a second electron to the quinone complex, so that the final acceptor quinone QB, upon double reduction and protonation, is released and substituted by a new quinone molecule from an exogenous pool (Feher et al. 1989). This photocycle can be reconstituted in solution and driven as long as the exogenous pools of either the external electron donor or acceptor run out (Milano et al. 2007). In these conditions, a sequence of flashes generates the so-called semiquinone oscillations because of the appearance of semiquinone at odd flashes and its disappearance at even flashes in the form of ubiquinol. The resulting flash-induced rise and drop of absorb− ance at 450 nm, where the reduced species (DQ− A, DQA QB − and DQA QB) absorb with a similar extinction coefficient (Kleinfeld et al. 1985), are typical spectroscopic behaviors of ubiquinone-based RCs and represent a valuable source of information on the kinetics and thermodynamics of ubiquinone reactions within these proteins (Vermeglio 1977; Wraight 1977). The first quantitative interpretation of absorbance changes was conducted on data obtained in the presence of a suitable electron donor, ensuring the complete D+ re-reduction before any charge recombination, and with excess ubiquinone concentration (Kleinfeld et al. 1984). Oscillations were damped since, at each step, a fraction 1/(1 + KE) of RCs is found in the inactive state DQ− A QB. Under these conditions, mutual linear relationships exist between the concentrations of different RC redox states at consecutive flashes, and the relevant system of linear equations can be solved analytically (Kleinfeld et al. 1984). In
13
order to properly fit the experimental data, the only adjustable parameters are the amplitude of semiquinone absorption after the first flash (ΔA1) and the interquinone electron transfer equilibrium constant KE according to the equation: m KE 1 + 2KE m �A1 1 − (−1) �Am = (4) 1 + KE 1 + KE where m is the flash number. This approach presents some limitations: • It is not suitable for improving the accuracy of the KE determination exploiting further information such as the different molar extinction coefficients of charged and uncharged species that are either available in the literature or can be inferred from ad hoc experiments. The above-mentioned procedure completely disregards this kind of information, possibly affecting the obtained KE value. • The flash sequence technique can also be exploited to obtain other parameters such as the quinone binding constant. However, to this end, one has to work with quinone concentrations leading to partial occupation of the available QB sites (subsaturating condition). • Various effects inherent to the experimental setup (excitation from the measuring light, quinole reoxidation, subsaturating flash light) appear in the recorded traces, which can strongly deviate from the ideal law described above. Disregarding these effects also can affect the meaning of the fitting parameters. In recent years, more advanced mathematical models for describing the oscillations also in the presence of inhibitors have been proposed (Halmschlager et al. 2002). In the present article, a more general kinetic model proposed by Shinkarev (1998) is implemented in a C-code used for numerical simulations of experimental data collected under sub-saturating ubiquinone concentrations. This condition induces a nonlinear oscillation pattern and enables evaluating the ubiquinone binding constant KQ, a parameter inaccessible working with saturated QB sites. Interestingly, unlike in the standard above-mentioned titration procedures, the use of the oscillation pattern does not impose depleting the QB site in the case of the native ubiquinone-10. In fact, 70–80 % of the QB sites are typically occupied in pristine RCs, which means that they are close to saturation in the CR essay. Conversely, oscillations lead naturally to a depletion of the QB sites and the relevant pattern changes even starting from completely filled QB sites. The advantage of the oscillation method resides hence in the possibility of avoiding harsh treatments of the protein, which may scramble the QB pocket. The binding constant KQ for UQ10 obtained in this article is hence the first, to our knowledge, obtained in a pristine QB site.
Eur Biophys J
Materials Chemicals All chemicals used in this work were purchased from Sigma-Aldrich and used without any further purification. Ubiquinone 0 (UQ0, 2,3-dimethoxy-5-methyl-1, 4-p-benzoubiquinone) stock solution was 40 mM in water. Ubiquinone 10 (UQ10) stock solution was 600 μM in 0.3 % Triton X-100 (TX). Ferrocene–methanol (FcnMeOH) stock solution was 10 mM in ethanol. Ferrocyanide (FeCy) stock solution was 100 mM in water. The pH-indicator absorption dye o-cresol red stock solution was 1 mM ethanol. All experiments were performed with freshly prepared solutions. Reaction centers RCs were isolated from blue-green strain R26 of the photosynthetic bacterium Rhodobacter (R.) sphaeroides following an established procedure using lauryldimethylamineN-oxide (LDAO) for extraction and ammonium sulfate precipitation and diethylaminoethyl (DEAE) chromatography for purification (Milano et al. 2009). The final ratio of absorbances at 280 and 802 nm was lower than 1.3 (optimal value for pure RC is 1.28), and the RC concentration was determined from the steady-state absorption at 802 nm using 288 mM−1 cm−1 as extinction coefficient. LDAO was exchanged with TX by overnight dialysis against 10 mM Tris buffer (pH 8.0), TX 0.03 % at 4 °C. The native UQ10 in the QB site was extracted with 1 % LDAO and 2 mM orthophenanthroline (Okamura et al. 1975). Steady‑state and transient optical spectroscopy The steady-state optical spectra were recorded by a Cary 5000 (Agilent) UV-visible-NIR spectrophotometer. Flashinduced absorbance changes were recorded using a kinetic spectrometer of local design described elsewhere (Milano et al. 2003).
Scheme 1 A general model of processes activated when reaction centers are subjected to light flashes in the presence of a fast electron donor. The waved arrows indicate processes happening upon flash excitation, double arrows denote fast chemical equilibria, and single arrows denote slower processes observable during the interval between two flashes
buffered sample (Maróti and Wraight 1988). The relevant data are presented in the supplementary material. Mathematical methods and fitting procedures Scheme 1 has been implemented in a C-code from the flow chart in figure S1 and used for generating simulated data sets and for fitting the relevant experimental data. The C-code, provided in a separate file, is described in the supplementary material along with additional details.
Results and discussion Modeling the oscillation pattern
Proton release measurements The pH indicator dye o-cresol red (7.5
