TIME-RESOLVED FTIR SPECTROSCOPY OF QUINONES IN RHODOBACTER-SPHAEROIDES REACTION CENTERS

Light-induced intermediates in reaction centers of Rhodobacter sphaeroides have been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy with a time-resolution of 25 ms at a spectral resolution of 4 cm-1. Following photoexcitation, an electron is rapidly transferred from the primary donor (P) to acceptor quinones, Q(A) and Q(B). Based on the different recombination lifetimes of the photoinduced intermediates (60-100 ms for P+Q(A)- --> PQ(A) and a few seconds for P+Q(A)Q(B)- --> PQ(A)Q(B)), a comparison between the P+Q(A)-(-)minus-PQ(A) (P+Q(A)-/PQ(A) and P+Q(A)Q(B)-(-)minus-PQ(A)Q(B) (P+Q(A)Q(B)-/PQ(A)Q(B)) spectra becomes reliable, since both difference spectra are measured: (i) for the same sample, (ii) at the same temperature, (iii) in the same chemical environment, (iv) from the same actinic event, and (v) with the same background. For the first time, the small variations observed between the two difference spectra, P+Q(A)-/PQ(A) and P+Q(A)Q(B)/PQ(A)Q(B), have been interpreted in terms of contributions from only Q(A), Q(B)-, Q(A)-, Q(B) and their amino acid partners without the interference from P and P+. Vibrational modes ascribed to neutral quinone carbonyls could not be singled out, instead several bands were related to changes in interaction of amino acid residues with Q(A) and Q(B) following photoexcitation. In particular, three bands (1670 cm-1, 1652 cm-1, 1630 cm-1) insensitive to H-1-H-2 exchange have been identified. The feature at 1670 cm-1 was not apparent in previous steady-state studies, the 1652-1 band has been associated to a conformational change of the peptide C = O of the conserved Ala M260 residue in the Q(A) pocket. The 1493 cm-1, 1480 cm-1 (Q(B)-) and the 1460 cm-1 (Q(A)-) bands have been revealed and attributed to C-C vibrations of the semiquinone anion without excluding the possibility of some C-O- contributions. The 1732 cm-1, 1555 cm-1, and 1533 cm-1 bands can be assigned to amino acid vibrations. The band at 1555 cm-1 could reflect the effect of the Q(A) delocalized charge on the ring of Trp M252 in van der Waals contact with Q(A).