Investigation of ubiquinol formation in isolated photosynthetic reaction centers by rapid-scan Fourier transform IR spectroscopy

Abstract

Light-induced formation of ubiquinol-10 in Rhodobacter sphaeroides reaction centers was followed by rapid-scan Fourier transform IR difference spectroscopy, a technique that allows the course of the reaction to be monitored, providing simultaneously information on the redox states of cofactors and on protein response. The spectrum recorded between 4 and 29 ms after the second flash showed bands at 1,470 and 1,707 cm⁻¹, possibly due to a QH⁻ intermediate state. Spectra recorded at longer delay times showed a different shape, with bands at 1,388 (+) and 1,433 (+) cm⁻¹ characteristic of ubiquinol [Mezzetti et al. FEBS Lett. 537:161–165 (2003)]. These spectra reflect the location of the ubiquinol molecule outside the Q_B binding site. This was confirmed by Fourier transform IR difference spectra recorded during and after continuous illumination in the presence of an excess of exogenous ubiquinone molecules, which revealed the process of ubiquinol formation, of ubiquinone/ubiquinol exchange at the Q_B site and between detergent micelles, and of Q_B⁻ and QH₂ reoxidation by external redox mediators. Kinetics analysis of the IR bands allowed us to estimate the ubiquinone/ubiquinol exchange rate between detergent micelles to approximately 1 s. The reoxidation rate of Q_B⁻ by external donors was found to be much lower than that of QH₂, most probably reflecting a stabilizing/protecting effect of the protein for the semiquinone form. A transient band at 1,707 cm⁻¹ observed in the first scan (4–29 ms) after both the first and the second flash possibly reflects transient protonation of the side chain of a carboxylic amino acid involved in proton transfer from the cytoplasm towards the Q_B site.

Appendix: kinetics model for continuous light excitation

The overall dynamics of light-induced reactions under and after photoaccumulation conditions is the results of a series of reactions and physicochemical processes, which have been reported in the literature (Okamura et al. 2000; Shinkarev and Wraight 1997, and references therein). To describe the experimental observations it was found necessary to consider a model which consists of 12 different states connected by 18 different reactions.

As stated in the text, an essential feature of the kinetics model is the slow exchange of ubiquinone and ubiquinol molecules between different LDAO micelles (Shinkarev and Wraight 1997); see also Fig. 8. The introduction of this slow exchange in the model was necessary to account for some experimental evidence (early Q_A⁻ formation under continuous illumination, slow electron transfer between Q_A⁻ and Q_B after switching off the lamp).

The reactions taken into account are listed in the following, with reactions 12 and 13 describing ubiquinone/ubiquinol exchange between micelles. The symbols used are those defined in the text and in addition the following: QH₂ (Q), ubiquinol (ubiquinone) in the same detergent micelle as the RC; ZH₂ (Z), ubiquinol (ubiquinone) in a detergent micelle, which does not contain a RC.

1	RCQA+Q=RCQAQB	Binding equilibrium for Q in the QB binding site
2	RCQAQB+light→RCQA −QB	Light-induced reduction of QA in a RC containing QB in its oxidized state
3	RCQAQB −+light→RCQA −QB −	Light-induced reduction of QA in a RC already in a QB − state
4	RCQAQBH2=RCQA+QH2	Dissociation of ubiquinol from the RC
5	RCQA+light→RCQA −	Light-induced reduction of QA in a RC with an empty QB site
6	QH2→Q	Reoxidation of ubiquinol in a RC-containing detergent micelle by external acceptors
7	RCQA −+Q=RCQA −QB	Binding equilibrium for Q in a RC with QA in its reduced state
8	RQAQB −→RQAQB	Reoxidation of QB − by external acceptors
9	RCQAQBH2+light→RQA −QBH2	Light-induced reduction of QA in a RC containing an ubiquinol bound to the QB site
10	RCQA −QBH2=QH2+RCQA −	Dissociation of ubiquinol from a RC with QA in its reduced state
11	RQAQBH2→RQAQB	Reoxidation of ubiquinol bound to a RC by external acceptors
12	Z=Q	Ubiquinone exchange between RC-containing micelles and pure detergent micelles
13	ZH2=QH2	Ubiquinol exchange between RC-containing micelles and pure detergent micelles
14	ZH2→Z	Ubiquinol reoxidation in pure detergent micelles
15	RCQA −QB=RCQAQB −	Electron transfer reaction between QA − and QB
16	RCQA −QB −=RCQA −QBH−	Electron transfer reaction between QA − and QB −
17	RCQA −→RCQA	Reoxidation of QA − by external acceptors
18	RCQAQBH−=RQAQBH2	Ubiquinol formation from QBH−

The kinetics evolution of IR bands characteristic of Q_A⁻, Q_B⁻, and QH₂ has been simulated with good accuracy (Figs. 6, 7) using rate constants and equilibrium constants from the literature (Okamura et al. 2000; McPherson et al. 1989; Shinkarev and Wraight 1997; Sebban et al. 1995 and references therein).⁷ The model was found to be not very sensitive to variations in the parameter values concerning chemical reactions and substrate binding equilibria. In contrast, the ubiquinone/ubiquinol exchange rate among pure detergent micelles and RC-containing micelles turned out to be a critical parameter in shaping the kinetics profiles of transient concentrations of the chemical species involved. Indeed, these transient concentrations are very sensitive to even relatively small variations in the kinetics constants for this exchange. This permitted the rate of this exchange to be estimated to 0.5–2 s.