ORIGINAL PAPER Ubiquinol formation in isolated photosynthetic reaction centres monitored by time-resolved differential FTIR in combination with 2D correlation spectroscopy and multivariate curve resolution Alberto Mezzetti & Lionel Blanchet & Anna de Juan & Winfried Leibl & Cyril Ruckebusch Received: 30 July 2010 / Revised: 7 October 2010 / Accepted: 10 October 2010 / Published online: 9 November 2010 # Springer-Verlag 2010 Abstract 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 -1 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 contri- butions. Three of them show marker bands for a mono- electronic reduction of the primary quinone Q A (Q A - /Q A transition, first contribution), for a monoelectronic reduc- tion of a secondary quinone Q B (Q B - /Q B transition, second contribution) and for ubiquinol formation (third contribu- tion). 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. Keywords Chemometrics . Multivariate curve resolution . 2D correlation spectroscopy . Rapid-scan FTIR . Purple bacteria Introduction Proton and electron transfer reactions are fundamental processes in biochemistry [1–3], and in bioenergetic systems they are often linked to quinone redox chemistry. Unlike most other enzymes, the reactions in photosynthetic reaction centres (RCs) can be easily triggered by light, and therefore, these RCs become good systems to investigate the general principles governing the kinetics and the Published in the special issue Chemometrics (VII Colloquium Chemiometricum Mediterraneum) with Guest Editors Marcelo Blanco, Juan M. Bosque-Sendra and Luis Cuadros-Rodríguez. Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4325-0) contains supplementary material, which is available to authorized users. A. Mezzetti : C. Ruckebusch (*) LASIR CNRS UMR 8516, Université Lille1 Sciences et Technologies, 59655 Villeneuve d’Ascq, France e-mail: cyril.ruckebusch@univ-lille1.fr A. Mezzetti : W. Leibl LPB, Service de Bioénergétique, Biologie Structurale et Mécanismes, URA CNRS 2096, IBITec-S, CEA-Saclay, Bat 532, 91191 Gif-sur-Yvette cedex, France L. Blanchet Institute for Molecules and Materials, Department of Analytical Chemistry, Radboud University Nijmegen, 6525 HP Nijmegen, The Netherlands A. de Juan Department of Analytical Chemistry, Universitat de Barcelona, 08007 Barcelona, Spain Anal Bioanal Chem (2011) 399:1999–2014 DOI 10.1007/s00216-010-4325-0