Spectral and thermodynamic characterization of cytochromes b in stroma vesicles from spinach chloroplasts

BAROLI, IRENE; FERNÁNDEZ-VELASCO, JAVIER G.; CROFTS, ANTONY R.

San Francisco, EEUU

Congreso; 35th Annual Meeting of the Biophysical Society; 1991

Biophysical Society

PS I enriched vesicles prepared by digitonin treatment of spinach chloroplasts have been critically studied in terms of their content of cytochrome b. In contrast with previous results (Peters et al, BBA 722:460-470,1983), cytochrome b6 showed both a spectral and thermodynamic heterogeneity (b-564, Em,7= -170 ± 10mV and b-563, Em,7 = -50 ± 10 mV) and on the other hand we found that the cytochrome described as b-559 (Em,7 = 32 mV) has actually its alpha peak at 560 nm and an Em, 7 = 50 ± 10 mV. No considerable amounts of either low (Em, 7 = 120 mV) or high potential (Em,7 = 350 mV) cytochrome b-559 could be detected. The stoichiometry b-564:b-563:b-560:b-559L:b-559 in PS I enriched vesicles was 1: 1: 1. 4: <0.1: <0.1. This is to be compared with that of 1:1:0.6:0.4:0.6 found in the PS II enriched membranes ("grana" recovered after digitonin treatment). These results are in agreement with the thermodynamic heterogeneity of the b-type cytochromes in the purified b6f complex (Hurt and Hauska, FEBS Lett. 153:413-419, 1983) and both the spectral heterogeneity of cytochrome b6 at room temperature (alpha peaks at 564 and 563 nm) and the presence of a cytochrome b-560 reported by Kramer and Crofts in intact chloroplasts (Kramer and Crofts in Balscheffsky ed. Cur. Res. Photosynth. 3,pp 381-384, Kluwer; Kramer, OM., Ph. D. Thesis UIUC/1990). We conclude that cyt b-564 and b-563 are uniformly distributed in the thylakoid system, cyt b-560 is enriched in the stroma vesicles and cyt b-559 (low and high potential forms) is enriched in the "grana" fraction.