Patrick V. Warren

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.

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.

Polypeptide composition of the photosystem I complex and the photosystem I core protein from Synechococcus sp. PCC 6301

The polypeptide composition of the Photosystem I complex from Synechococcus sp. PCC 6301 was determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. The PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaK and PsaL proteins, as well as three polypeptides with apparent masses less than 8 kDa and small amounts of the 12.6 kDa GlnB (PII) protein, wee present in the Photosystem I complex. No proteins homologous to the PsaG and PsaH subunits of eukaryotic Photosystem I complexes were detected. When the Photosystem I complex was treated with 6.8 M urea and ultrafiltered using a 100 kDa cutoff membrane, the resulting Photosystem I core protein was found to be depleted of the PsaC, PsaD and PsaE proteins. The filtrate contained the missing proteins, along with five proteolytically-cleaved polypeptides with apparent masses of less than 16 kDa and with N-termini identical to that of the PsaD protein. The PsaF and PsaL proteins, along with the three less than 8 kDa polypeptides, were not released from the Photosystem I complex to any significant extent, but low-abundance polypeptides with N-termini identical to those of PsaF and PsaL were found in the filtrate with apparent masses slightly smaller than those found in the native Photosystem I complex. When the filtrate was incubated with FeCl3, Na2S and beta-mercaptoethanol in the presence of the isolated Photosystem I core protein, the PsaC, PsaD and PsaE proteins were rebound to reconstitute a Photosystem I complex functional in light-induced electron flow from P700 to FA/FB. In the absence of the iron-sulfur reconstitution agents, there was little rebinding of the PsaC, psaD or PsaE proteins to the Photosystem I core protein. No binding of the truncated PsaD polypeptides occurred, either in the presence or absence of the iron-sulfur reagents. The reconstitution of the FA/FB iron-sulfur clusters thus appears to be a necessary precondition for rebinding of the PsaC, psaD and psaE proteins to the Photosystem I core protein.

Site-directed conversion of cysteine-565 to serine in PsaB of photosystem I results in the assembly of iron-sulfur [3Fe-4S] and iron-sulfur [4Fe-4S] clusters in Fx. A mixed-ligand iron-sulfur [4Fe-4S] cluster is capable of electron transfer to FA and FB

We reported earlier [Smart, L. B., Warren, P. V., Golbeck, J. H., & McIntosh, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1132-1136] that the site-directed conversion of cysteine-565 to serine (C565S) in PsaB of Synechocystis sp. PCC 6803 leads to an accumulation of photosystem I polypeptides and the low-temperature photoreduction of the terminal electron acceptors FA and FB. In this paper, we report the occurrence of a [3Fe-4S]1 + ,0 cluster in dodecyl maltoside-solubilized photosystem I complexes prepared from the C565S mutant. The [3Fe-4S] cluster is reducible with dithionite at pH 6.5, implying a midpoint potential considerably more oxidizing than either FA or FB. Similar to the behavior of FX, the [3Fe-4S] cluster undergoes partial, reversible photoreduction when the complex is illuminated at 15 K, and complete photoreduction when the sample is illuminated during freezing. Contrary to the result expected in the presence of a relatively high-potential FX, there is significant low-temperature and room temperature photoreduction of FA and FB in the C565S complex. Although the FA and FB resonances are more intense when the complex is frozen during illumination, they still account for < 60% of FA and FB found by chemical reduction. When the FA and FB clusters are prereduced with dithionite at pH 10.0, a new set of resonances appear upon illumination at g = 2.015, 1.941, and 1.811, and disappear on subsequent darkness. The species giving rise to this signal is most likely a mixed-ligand [4Fe-4S]2+,1+ cluster located in the FX site.(ABSTRACT TRUNCATED AT 250 WORDS)