Identification of a Ubiquinone-Ubiquinol Quinhydrone Complex in Bacterial Photosynthetic Membranes and Isolated Reaction Centers by Time-Resolved Infrared Spectroscopy

Abstract

Ubiquinone redox chemistry is of fundamental importance in biochemistry, notably in bioenergetics. The bi-electronic reduction of ubiquinone to ubiquinol has been widely studied, including by Fourier transform infrared (FTIR) difference spectroscopy, in several systems. In this paper, we have recorded static and time-resolved FTIR difference spectra reflecting light-induced ubiquinone reduction to ubiquinol in bacterial photosynthetic membranes and in detergent-isolated photosynthetic bacterial reaction centers. We found compelling evidence that in both systems under strong light illumination-and also in detergent-isolated reaction centers after two saturating flashes-a ubiquinone-ubiquinol charge-transfer quinhydrone complex, characterized by a characteristic band at ~1565 cm^-1, can be formed. Quantum chemistry calculations confirmed that such a band is due to formation of a quinhydrone complex. We propose that the formation of such a complex takes place when Q and QH₂ are forced, by spatial constraints, to share a common limited space as, for instance, in detergent micelles, or when an incoming quinone from the pool meets, in the channel for quinone/quinol exchange at the Q_B site, a quinol coming out. This latter situation can take place both in isolated and membrane bound reaction centers Possible consequences of the formation of this charge-transfer complex under physiological conditions are discussed.

Keywords: Fourier Transform Infrared (FTIR) difference spectroscopy; Rhodobacter (Rb.) sphaeroides; bacterial reaction center (RC); charge-transfer complex; chromatophores; quinhydrone; rapid-scan FTIR; ubiquinol; ubiquinone.

Figure 1

(a) Ubiquinone structural formula and its reduction to ubiquinol. (b) Scheme of ubiquinone photoreduction in photosynthetic membranes. After 2 consecutive series of photoinduced electron transfer reactions, the doubly reduced Q_BH₂ leaves the RC and is replaced by a new Q molecule coming from the quinone pool in the membrane. (c) Scheme if ubiquinone photoreduction in detergent-isolated photosynthetic reaction centers (RCs). In this case, after 2 consecutive series of photoinduced electron transfer reactions, the doubly reduced Q_BH₂ leaves the RC to go into the detergent phase of the RC-containing detergent micelle. A Q/QH₂ exchange at the Q_B site also takes place in this case. However, here the situation is complicated by the Q/QH₂ exchange between RC-containing detergent micelles and pure detergent micelles. P stands for the primary donor (dimer of BChl a molecules); ED stands for a generic electron donor, in the case of the experiments presented in this manuscript 2,3,5,6-tetramethyl-p-phenylenediamine (diaminodurene DAD).

Figure 2

Fourier Transform Infrared (FTIR) difference spectra on Rhoodobacter (Rb.) sphaeroides chromatophores recorded under illumination with different intensities, between 2 and 4 s after the onset of light exposure. The relative light intensity is (top to bottom) 1 (green):2 (blue):5 (purple):8 (red). Spectra are recorded on the same sample; measurement times (both in terms of light exposure and number of measurement cycles) are minimized in order to have negligeable light-induced sample degradation or fatigue. Experiments repeated on other samples provided similar results. Peak position should be considered to have a ±1 cm⁻¹ uncertainty.

Figure 3

Time evolution of the FTIR difference spectra of Rb. sphaeroides chromatophores after onset of continuous illumination at higher illumination intensity (upper two traces) and after switching off the illumination (two traces at the bottom). Black trace (first from top): recorded between 0 and 434 ms after light onset. Red trace (second from top): recorded between 3878 and 4312 ms after light onset. Green trace (third from top): recorded between 434 and 868 ms after switching off the light. Blue trace (fourth from top) recorded between 1300 and 3000 ms after switching off the light. Peak position should be considered to have a ±1 cm⁻¹ uncertainty.

Figure 4

FTIR difference spectra reflecting reduction of the quinone pool with formation of the new species characterized by a band at ~1565 cm⁻¹. From top to bottom: green spectrum, taken from [7], obtained after a Multivariate Curve Resolution treatment of time-resolved FTIR difference data on chromatophores. Blue spectrum, recorded between 1, 3 and 3 s after strong illumination (last bottom trace of time-resolved FTIR spectra shown in Figure 3). Black spectrum: spectrum recorded on chromatophores between 1 and 3 s after strong illumination in D₂O. Peak position should be considered to have a ±1 cm⁻¹ uncertainty. Green spectrum was adapted with permission from Ref. [7], Copyright 2009 American Chemical Society.

Figure 5

FTIR difference spectra recorded after one (red trace—2nd from top) and two flashes (black trace—1st from top). The difference spectrum after 2 flashes represents a mixture of ~75% QH₂ state and of 25% Q_B⁻ state [6]. The third trace from the top (green) was obtained by subtracting (multiplied by a 0.3 coefficient) a Q_B⁻/Q_B difference spectrum (red—2nd trace from the top) from the difference spectrum obtained after two flashes (black—1st trace from the top). This third trace represents the species formed after a double reduction of Q_B and shows an intense positive 1564 cm⁻¹ band. The fourth trace from the top (black) was taken from [15], obtained after a chemometric analysis of time-resolved FTIR difference spectra of isolated RCs under and after continuous illumination, where four spectral contributions were identified. The plotted trace—characterized by a strong positive peak at 1564 cm⁻¹—is believed to represent a double-reduced form of Q [15]. The last trace from the top (black) was reprinted by permission from Ref. [15], Copyright 2011, Springer.

Figure 6

Upper trace, black: FTIR difference spectrum recorded on chromatophores between 1302 and 1736 ms after a 4.3 s period of intense illumination (see also Figure 3). Lower trace (green): calculated FTIR spectrum in detergent-isolated RCs after two saturation flashes (see also Figure 5). Inset: spectra of synthetic Q₆H₂ in cyclohexane in freshly prepared solution (black trace) and after exposure of the solution to air for some hours (red trace).

Figure 7

Model quinone molecule used for theoretical calculations of IR frequencies.

Figure 8

Stable quinone–quinol quinhydrone complex after optimizations. (a) Most stable geometry (∆_rE = 106 kJ mol⁻¹); (b) stable complex with two hydrogen bonds (∆_rE = 90 kJ mol⁻¹).

Figure 9

Conditions under which quinhydrone complex could form in chromatophores (a) or detergent-isolated RCs (b).