Isolation of the intact photosystem I reaction center core containing P700 and iron‐sulfur center F_X

Abstract

The photosystem I reaction center core containing P700 and iron‐sulfur center FX has been isolated from a Synechococcus photosystem I particle with 6.8 M urea at pH 10.0 followed by sucrose density ultracentrifugation. The reaction center core has retained > 90% of FX and 100% of P700 (determined by optical spectroscopy) but is totally devoid of iron‐sulfur centers FA and FB (determined by optical and ESR spectroscopy). SDS‐PAGE indicates the retention of the 57 kDa reaction center polypeptide(s) but the total absence of the 16.4 and 8.1 kDa polypeptides. The loss of FA and FB is further reflected in the decline of acid‐labile sulfide from 11.8 ± 0.4 S2− /P700 in the control particle to 4.6 ± 0.3 S2− /P700 in the reaction center core. This preparation represents the first isolation of an intact reaction center core incorporating the components P700 and FX but totally lacking FA and FB.

Identification of photosystem I components from the cyanobacterium, Synechococcus vulcanus by N‐terminal sequencing

“…Note that the 9.5 kDa band is very weak and diffuse, in agreement with the known properties of higher plant iron-sulfur proteins [28]. Since the 6.5, 5 and 4.1 kDa proteins are not resolved by conventional SDS-PAGE (not shown), this separation profile would be basically the same as those reported for other cyanobacterial PSI complexes [3][4][5][6]. For example, Synechococcus PCC 6301 PS I complex has been reported to contain 18, 17.7, 16 and 10 kDa with a molar ratio of 0.7: 1.0:0.5:1.6 [3], which seem to correspond to our 18, 14, 12 and 9 kDa components with a compatible ratio.…”

“…However, direct identification of the protein was difficult probably because this protein is so poorly stained on the gel: the iron-sulfur protein has been overlooked on the gel in spite of its abundance in the PSI complex. On the other hand, four low-molecular-mass proteins have been typically resolved in cyanobacteria by conventional SDS-PAGE, and their characterization and correspondence have been sporadically reported [3][4][5][6]21,22]. In this study, we resolved eight proteins and showed more clearly the correspondence between the 18, 14, 12 and 9 kDa proteins of S.…”

“…The presence of this protein in the cyanobacterial PSI complex as well as in thylakoid membranes has been suggested by kinetic analysis [5,6,[12][13][14], detection of nonheme iron-sulfur centers A and B by low temperature EPR [3,5,6] and 35S labeling of Cys residues [34]. However, direct identification of the protein was difficult probably because this protein is so poorly stained on the gel: the iron-sulfur protein has been overlooked on the gel in spite of its abundance in the PSI complex.…”

“…In higher plants, the PSI complex consists of three groups of protein components: (i) two CP I apoproteins (psaA and psaB gene products) bearing PT00, chlorophylls and a non-heine iron-sulfur center X; (ii) several components of 8 to 22 kDa which belong to the PS I core complex; (iii) several LHC I components of 20-25 kDa [1,2]. Similarly, cyanobacterial PSI complex has been reported to contain four components of 8 to Abbreviations: CP I, photosystem I chlorophyll-protein; EPR, electron paramagnetic resonance; LHC I, light-harvesting chlorophyll-protein complex associated with photosystem I; ORF, open reading frame; PS 1 and II, photosystem I and II, respectively; PVDF, polyvinylidene difluoride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis 20 kDa and the two CP I apoproteins [3][4][5][6]. Since the cyanobacterial complex does not carry any LHC I-type components, these low-molecularmass components might have structural or functional roles similar to the core components of higher plants.…”

Interaction between Photosystem I and Flavodoxin from the Cyanobacterium Synechococcus sp. PCC 7002 as Revealed by Chemical Cross‐Linking

“…PS 1 was purified from the wild-type strain PR-6000, a psaE mutant strain, PR6302, , and a psaL mutant strain, PR6309 (Schluchter, 1994), of the marine cyanobacterium Synechococcus sp. PCC 7002, essentially as described previously (Golbeck et al, 1988). Recombinant flavodoxin of the same organism was expressed in Escherichia coli strain BL21 harboring isiB (Leonhardt and Straus, 1992) in the expression vector pSE280 (Brosius, 1989) and purified esseiitially as dcscribed (Fillat et al, 1991).…”