Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site

Abstract

Time–resolved step-scan Fourier transform infrared difference spectroscopy has been used to study cyanobacterial photosystem I photosynthetic reaction centers from Synechocystis sp. PCC 6803 (S6803) with four high-potential, 1,4-naphthoquinones incorporated into the A1 binding site. The high-potential naphthoquinones are 2-chloro-, 2-bromo-, 2,3-dichloro- and 2,3-dibromo-1,4-naphthoquinone. “Foreign minus native” double difference spectra (DDS) were constructed by subtracting difference spectra for native photosystem I (with phylloquinone in the A1 binding site) from corresponding spectra obtained using photosystem I with the different quinones incorporated. To help assess and assign bands in the difference and double difference spectra, density functional theory based vibrational frequency calculations for the different quinones in solvent, or in the presence of a single asymmetric H– bond to either a water molecule or a peptide backbone NH group, were undertaken. Calculated and experimental spectra agree best for the peptide backbone asymmetrically H– bonded system. By comparing multiple sets of double difference spectra, several new bands for the native quinone (phylloquinone) are identified. By comparing calculated and experimental spectra we conclude that the mono-substituted halogenated NQs can occupy the binding site in either of two different orientations, with the chlorine or bromine atom being either ortho or meta to the H– bonded CO group.