Relative enrichment of P-870 in photosynthetic reaction centers treated with sodium borohydride

Abstract

Reaction centers from Rhodopseudomonas sphaeroides R26 in 0.03% LDAO/0.1 M Tris (initial pH = 8.0) were treated with sodium borohydride. The pH of the reaction center solution was never allowed to exceed 10. Absorption spectra taken at various times show that for approx. 8 h after the first addition of NaBH 4, A 865 (P-870) and A 760 diminish very little (no more than 15% loss each), while A 800 diminishes markedly (45% loss) and a new peak (at 715 nm) grows in at approximately the same rate that A 800 decays. Separate experiments on the absorption and 1H-NMR spectra of purified bacteriochlorophyll (BChl) and bacteriopheophytin (BPh), and their respective NaBH 4-reduction products, reveal that A 715 in NaBH 4-treated reaction centers most likely results from 2a-deoxy-2a-(hydroxy)BPh a, a BPh reduction product. We conclude that at least part of the BChl contributing to A 800 in the reaction center is reduced at the acetyl group by NaBH 4, apparently with concomitant pheophytinization; if two molecules of BChl contribute equally to that absorption, one of them is reduced. Thus, it is plausible that, of the 6 bacteriochlorin molecules in the reaction center, only one is so configured that its acetyl group is both accessible to the solvent and reactive. In addition to providing a new geometric marker for reaction centers, the NaBH 4-reduction technique should make it possible to decide whether any small spectral change in the 800-nm region can be ruled out as the higher-energy exciton transition of the putative BChl special pair. The technique should be interesting to apply to reaction centers from other organisms, especially Rhodopseudomonas viridis. Because NaBH 4 treatment is unlikely to significantly modify protein structure, investigations of the photochemical properties of the modified reaction centers should be highly informative, especially since reversible bleaching of P-870 in NaBH 4-treated reaction centers now has been observed (Maroti, P., Wraight, C.A. and Pearlstein, R.M., unpublished results).