Efficient conditions for the photoaccumulation of H-A(-) in the photosynthetic reaction centre of Rhodobacter sphaeroides R26 with uniformly labelledbacteriopheophytin monitored by Fourier transform infrared difference spectroscopy

The native bacteriopheophytins (BPheo) a at sites H-A and H-B in the reacti on centre (RC) of Rhodobacter sphaeroides R26 have been exchanged with unif ormly C-13, N-15 labelled BPheo a. Exchange at the H-A site was > 50% as mo nitored with light-induced FTIR-difference spectroscopy indicated by the sh ift of the band at 1590 cm(-1). The photoreduction of H-A has been monitore d by light-induced FTIR-difference spectroscopy at 22 degrees C in the pres ence of reductant and mediator: either sodium dithionite and cytochrome c, or sodium dithionite and dyes (potassium indigotetrasulfonate, neutral red) . New experimental conditions are described for Ha photoaccumulation, and l ight-induced FTIR-difference spectra characteristic of H-A(-) are reported. The H-A(-)/H-A FTIR-difference spectra of Rb. sphaeroides R26 RCs in which uniformly C-13, N-15 labelled BPheo a replaces the photoactive BPheo will allow the BPheo modes to be assigned and to be discriminated from those of the RC protein. (C) 1999 Elsevier Science B.V. All rights reserved.