TOPOGRAPHY OF THE HEME PROSTHETIC GROUP OF CYTOCHROME B-559 IN THE PHOTOSYSTEM-II REACTION-CENTER

The topography of the heme prosthetic group of cytochrome b-559 of the photosystem II reaction center was determined from measurement of the orientation of its alpha- and beta-polypeptides in thylakoid membrane s of spinach chloroplasts and in osmotically disrupted cells of the cy anobacterium Synechocystis sp. PCC 6803. The accessibility to trypsin proteolysis of an epitope located near the solvent-exposed N-terminus of the beta-subunit was compared to that of the alpha-subunit, whose N - and C-termini had previously been localized from the trypsinolysis p attern to the stromal and lumenal sides of spinach thylakoid membranes , respectively [Tae et al. (1988) Biochemistry 27, 9075-9080; Vallon e t al. (1989) Biochim. Biophys. Acta 975, 132-141]. The N-terminal epit ope of the cyanobacterial beta-subunit was modified by introducing a t ridecapeptide epitope, previously found to be immunoreactive, from the C-terminal region of the spinach chloroplast alpha-subunit. This epit ope had no homology with the cyanobacterial alpha-subunit. The cells w ith the hybrid beta-subunit retained full photosynthetic activity. The intactness of membranes from osmotically shocked cyanobacteria was te sted by trypsin inaccessibility to (a) the alpha-subunit C-terminus an d (b) the manganese-stabilizing protein (MSP) of the oxygen-evolving c omplex that is on the lumenal side of the membrane. The loss after try psinolysis of most of the beta-subunit immunoreactivity, under conditi ons where (i) the alpha-subunit was cleaved near the N-terminus in bot h spinach thylakoids and osmotically shocked cyanobacterial membranes and (ii) the MSP protein in cyanobacteria was not disrupted, implied t hat the orientation of the beta-subunit was parallel to that of the al pha-subunit in both kinds of membranes. This is consistent with expect ations from the cis-positive rule for orientation of integral membrane proteins [von Heijne and Gavel (1988) fur. J. Biochem. 74, 671-678] a nd the very positive E(m) of cytochrome b-559 [Krishtalik et al. (1993 ) Biophys. J. 65, 184-195]. For this cytochrome, the polypeptide orien tation uniquely determines the heme topography, because its coordinati on is bis-histidine and each polypeptide contains only one histidine, located near the N-terminal side of the single hydrophobic alpha-helix . The heme prosthetic group must therefore be located close to the str omal or cytoplasmic interface of the membrane.