Jindong Zhao

Assembly of photosystem I. I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity.

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of theSynechocystis sp. PCC 6803 gene slr2034 andArabidopsis thaliana HCF136, and the psbEFLJoperon, was cloned from the cyanobacterium Synechococcussp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene ofSynechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was ∼80% of the wild-type level. RubA contains a domain of ∼50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane α-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain inEscherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type (∼40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Characterization of psal and psaL Mutants of Synechococcus sp. Strain PCC 7002: A New Model for State Transitions in Cyanobacteria

The psal and psaL genes were characterized from the cyanobacterium Synechococcus sp. strain PCC 7002. The gene organization was different from that reported for other cyanobacteria with psal occurring upstream and being divergently transcribed from the psaL gene. Mutants lacking Psal or PsaL were generated by interposon mutagenesis and characterized physiologically and biochemically. Mutant strains PR6307 (Δpsal−), PR6308 (psal) and PR6309 (psaL−) had doubling times similar to that of the wild type under both high‐ and low‐intensity white light, but all grew more slowly than the wild type in green light. Only monomeric photosystem I (PS I) complexes could be isolated from each mutant strain when Triton X‐100 was used to solubilize thylakoid membranes; however, approximately 10% of the PS I complexes from the psal mutants, but not the psaL mutant, could be isolated as trimers when n‐do‐decyl β‐D‐maltoside was used. Compositional analyses of the mutant PS I complexes indicate that the presence of PsaL is required for trimer formation or stabilization and that Psal plays a role in stabilizing the binding of both PsaL and PsaM to the PS I complex. Strain PR6309 (psaL−) was capable of performing a state 2 to state 1 transition approximately three times more rapidly than the wild type. Because the monomeric PS I complexes of this mutant should be capable of diffusing more rapidly than trimeric complexes, these data suggest that PS I complexes rather than phycobilisomes might move during state transitions. A “mobile‐PS I” model for state transitions that incorporates these ideas is discussed.

Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coli.

The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria.

Assembly of Photosystem I II. RUBREDOXIN IS REQUIRED FOR THE IN VIVO ASSEMBLY OF FX IN SYNECHOCOCCUS SP. PCC 7002 AS SHOWN BY OPTICAL AND EPR SPECTROSCOPY

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, FX, FA, and FB, are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubAmutant. The flash-induced decay kinetics of both P700+ in the visible and A1 − in the near-UV show that charge recombination occurs between P700+ and A1 − in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A1. In agreement, the spin-polarized X-band EPR spectrum of P700+ A1 − at low temperature shows that an electron cycle between A1 − and P700+ occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [FA/FB]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700+ to A1 − center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of FX, FB, and FA, the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the FX iron-sulfur cluster but that FX is not required for the biosynthesis of trimeric P700-A1 cores. Since the PsaC protein requires the presence of FX for binding, the absence of FA and FB may be an indirect result of the absence of FX.

Reconstitution of electron transport in photosystem I with PsaC and PsaD proteins expressed in Escherichia coli

A fusion protein, denoted PsaCl, which contains an amino‐terminal extension of five amino acids (MEHSM...) and is derived from an in vitro modified form of the psaC gene of Synechococcus sp. PCC 7002, has been over‐expressed in Escherichia coli. The product of the psaD gene of Nostoc sp. PCC 8009 has similarly been over‐expressed. The PsaCl and PsaD proteins can be combined with the photosystem I core protein of Synechococcus sp. PCC 6301 to reconstitute electron transport from P700 to the terminal FA/FB acceptors. Reconstitution was found to be absolutely dependent on reinsertion of the iron‐sulfur clusters in the PsaCI apoprotein and on the presence of the PsaD protein. This implies that the PsaCl holoprotein does not bind solely to the PsaA/PsaB heterodimer but rather that its interaction with these proteins is mediated through the PsaD protein.