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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 QB 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. [ABSTRACT FROM AUTHOR]

The PufX protein is found in the photosynthetic membranes of several purple bacteria and is involved in ubiquinol-ubiquinone exchange at the Q B site of the reaction center. We have studied quinone pool reduction in chromatophores from PufX+ and PufX− strains of Rhodobacter capsulatus by time-resolved FTIR difference spectroscopy under and after continuous illumination. To our knowledge, it is the first time that quinone pool reduction has been directly followed in real time in Rba. capsulatus membranes. Thanks to the availability in the literature of IR marker bands for protein conformational changes, ubiquinone consumption, ubiquinol production, Q---QH 2 quinhydrone complex formation, as well as for RC-bound Q A − and Q B − semiquinone species, it is possible to follow all the molecular events associated with light-induced quinone pool reduction. In Rba. capsulatus PufX + chromatophores, these events resemble the ones found in Rba. sphaeroides wild-type membranes. In PufX− chromatophores the situation is different. Spectra recorded during 22.7 s of illumination showed a much smaller amount of photoreduced quinol, consistent with previous observations that PufX is required for efficient QH 2 /Q exchange at the Q B site of the RC. Q consumption and QH 2 formation are rapidly associated with Q A − formation, showing that the structure of the RC-LH1 complex in PufX− membranes does not provide efficient access to the Q B site of the RC to a large fraction of the quinone pool, evidently because the LH1 ring increases in size to impair access to the RC. The presence of a positive band at 1560 cm−1 suggests also the transient formation, in a fraction of chromatophores or of RC-LH1 complexes, of a Q---QH 2 quinhydrone complex. Experiments carried out after 2-flash and 10-flash sequences make it possible to estimate that the size of the quinone pool with access to the Q B site in PufX− membranes is ≥ 5 ubiquinone molecules per RC. The results are discussed in the framework of the current knowledge of protein organization and quinone pool reduction in bacterial photosynthetic membranes. [Display omitted] • Quinone pool reduction was monitored by time-resolved FTIR spectroscopy in Rba. capsulatus PufX+ and PufX− chromatophores. • Protein conformational changes, formation of quinhydrone and of semiquinone species were observed by FTIR spectroscopy. • In PufX−membranes the RC-LH1 complex structure does not provide efficient access to the Q B site to all the quinone pool. • The size of the quinone pool with rapid access to the Q B site in PufX− membranes is ≥ 5 ubiquinone molecules per RC. [ABSTRACT FROM AUTHOR]

Light-induced infrared difference spectroscopy (IR-DS) has been used, especially in the last decade, to investigate early photophysics, energy transfer and photoprotection mechanisms in isolated and membrane-bound light harvesting complexes (LHCs). The technique has the definite advantage to give information on how the pigments and the other constituents of the biological system (proteins, membranes, etc.) evolve during a given photoreaction. Different static and time-resolved approaches have been used. Compared to the application of IR-DS to photosynthetic Reaction Centers (RCs), however, IR-DS applied to LHCs is still in an almost pioneering age: very often sophisticated techniques (step-scan FTIR, ultrafast IR) or data analysis strategies (global analysis, target analysis, multivariate curve resolution) are needed. In addition, band assignment is usually more complicated than in RCs. The results obtained on the studied systems (chromatophores and RC-LHC supercomplexes from purple bacteria; Peridinin-Chlorophyll-a-Proteins from dinoflagellates; isolated LHCII from plants; thylakoids; Orange Carotenoid Protein from cyanobacteria) are summarized. A description of the different IR-DS techniques used is also provided, and the most stimulating perspectives are also described. Especially if used synergically with other biophysical techniques, light-induced IR-DS represents an important tool in the investigation of photophysical/photochemical reactions in LHCs and LHC-containing systems.

Time-resolved (TR) infrared (IR) spectroscopy in the nanosecond to second timescale has been extensively used, in the last 30 years, in the study of photosynthetic systems. Interesting results have also been obtained at lower time resolution (minutes or even hours). In this review, we first describe the used techniques-dispersive IR, laser diode IR, rapid-scan Fourier transform (FT)IR, step-scan FTIR-underlying the advantages and disadvantages of each of them. Then, the main TR-IR results obtained so far in the investigation of photosynthetic reactions (in reaction centers, in light-harvesting systems, but also in entire membranes or even in living organisms) are presented. Finally, after the general conclusions, the perspectives in the field of TR-IR applied to photosynthesis are described. [ABSTRACT FROM AUTHOR]

Light-induced infrared difference spectroscopy (IR-DS) has been used, especially in the last decade, to investigate early photophysics, energy transfer and photoprotection mechanisms in isolated and membrane-bound light harvesting complexes (LHCs). The technique has the definite advantage to give information on how the pigments and the other constituents of the biological system (proteins, membranes, etc.) evolve during a given photoreaction. Different static and time-resolved approaches have been used. Compared to the application of IR-DS to photosynthetic Reaction Centers (RCs), however, IR-DS applied to LHCs is still in an almost pioneering age: very often sophisticated techniques (step-scan FTIR, ultrafast IR) or data analysis strategies (global analysis, target analysis, multivariate curve resolution) are needed. In addition, band assignment is usually more complicated than in RCs. The results obtained on the studied systems (chromatophores and RC-LHC supercomplexes from purple bacteria; Peridinin-Chlorophyll-a-Proteins from dinoflagellates; isolated LHCII from plants; thylakoids; Orange Carotenoid Protein from cyanobacteria) are summarized. A description of the different IR-DS techniques used is also provided, and the most stimulating perspectives are also described. Especially if used synergically with other biophysical techniques, light-induced IR-DS represents an important tool in the investigation of photophysical/photochemical reactions in LHCs and LHC-containing systems. [ABSTRACT FROM AUTHOR]

Two-dimensional correlation analysis was carried out in combination with multivariate curve resolution-alternating least squares (MCR-ALS) to analyse time-resolved infrared (IR) difference spectra probing photo-induced ubiquinol formation in detergent-isolated reaction centres from Rhodobacter sphaeroides. The dynamic 2D IR correlation spectra have not only allowed the determination of the concomitance or non-concomitance of different chemical events through known marker bands but also have helped identify new vibrational bands related to the complex series of photochemical and redox reactions. In particular, a strong positive band located at 1565 cm was found to be synchronous with the process of ubiquinol formation. In addition, a tailored MCR-ALS analysis was performed using a priori chemical knowledge of the system, in particular including the pure spectrum of one species obtained from an external measurement. Enhancing the MCR-ALS performance in this way in time-dependent processes is relevant, especially when other essential pieces of information, such as kinetic models, are unavailable. The results give evidence of four independent spectral contributions. Three of them show marker bands for a monoelectronic reduction of the primary quinone Q (Q/Q transition, first contribution), for a monoelectronic reduction of a secondary quinone Q (Q/Q transition, second contribution) and for ubiquinol formation (third contribution). The results obtained also confirm that a key rate-limiting factor is the slow ubiquinone and ubiquinol exchange among micelles, which strongly influences the kinetic profiles of the involved species. [ABSTRACT FROM AUTHOR]

Rapid-scan FTIR difference spectroscopy was used to investigate light-induced reduction of the ubiquinone QB, and the oxidation kinetics of QB- and QH2 by external mediators in isolated reaction centers (RCs) from R. sphaeroides. As redox mediators, a Ferrocyanide/N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) mixture and an Ascorbate/2,3,5,6-tetramethyl-p-phenylenediamine (DAD) mixture are compared. Results show that TMPDred rapidly reduces the photoproduced P870+ primary donor. The process is fast enough to record rapid-scan FTIR spectra devoid of P870+ bands down to 260 K. Results show also that TMPDox oxidises both QB- and QH2 faster than DADox. In particular, QB- is oxidised faster than QH2 at all temperatures studied. Results are discussed in the framework of time-resolved infrared studies on R. sphaeroides RCs, showing advantages/drawbacks of the proposed experimental approach. [ABSTRACT FROM AUTHOR]

Abstract: X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628–11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni–SIa reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni–SCO and Ni–SCOred. At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni–R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni–SCO state results in Ni–SIa that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol−1. In addition, formation of a paramagnetic CO-inhibited state (Ni–CO) was observed that results from the interaction of carbon monoxide with the Ni–L state. It is proposed that the nickel in Ni–CO is in a formal monovalent state (Ni1+). [Copyright &y& Elsevier]

Abstract: Rapid-scan FTIR difference spectroscopy was used to investigate proton and electron transfer reactions in photosynthetic reaction centers from Rhodobacter sphaeroides. Experiments at different temperatures and in the presence of D2O have provided strong indication that a transient band at 1707cm−1 previously identified after both the 1st and the 2nd flashes [A. Mezzetti, W. Leibl, Eur. Biophys. J. 34 (2005) 921] is given by a transient protonation of the side chain of a Asp or Glu residue situated on the proton transfer pathway from the cytoplasm to the QB site. Experiments in D2O on a Asp-M17→Asn mutant reaction center, where the proton and electron transfer reactions are slowed down compared to the wild-type, showed that the kinetic isotope effect induced by H/D exchange slows down the electron transfer reaction after the 1st flash, confirming the strong coupling between proton and electron transfer. Rapid-scan FTIR experiments on Cd2+-treated reaction centers, in agreement with previous UV–vis measurements [P. Adelroth, M.L. Paddock, L.B. Sagle, G. Feher, M.Y. Okamura, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 13086], showed that upon addition of Cd2+, which inhibits proton uptake, the QA −QB →QAQB − reaction is slowed. Interestingly, the transient 1707cm−1 band is not visible in the first spectrum recorded early after the flash. This strongly suggests its identification with a residue situated on the proton transfer pathway, which is perturbed upon metal cation binding. [Copyright &y& Elsevier]

Photosynthetic reaction centres and membranes are systems of particular interest and are often taken as models to investigate the molecular mechanisms of selected bioenergetic reactions. In this work, a multivariate curve resolution by alternating least squares procedure is detailed for resolution of time-resolved difference FTIR spectra probing the evolution of quinone reduction in photosynthetic membranes from Rhodobacter sphaeroides under photoexcitation. For this purpose, different data sets were acquired in the same time range and spectroscopic domain under slightly different experimental conditions. To enable resolution and provide meaningful results the different data sets were arranged in an augmented matrix. This strategy enabled recovery of three different species despite rank-deficiency conditions. It also results in better definition (identity and evolution) of the contributions. From the resolved spectra, the species have been attributed to: 1. the formation of ubiquinol, more precisely the disappearance of Q/appearance of QH2; 2. conformational change of the protein in the surrounding biological medium; 3. oxidation of diaminodurene, a redox mediator. Because, moreover, results obtained from augmented data sets strategies enable quantitative and qualitative interpretation of concentration profiles, other effects, for example the consequence of repeated light excitation of the same sample, choice of illumination power, or the number of spectra accumulated could be compared and discussed. [ABSTRACT FROM AUTHOR]