The two species of 5‐aminolevulinic acid synthetase were purified about 4200‐fold for fraction I and about 2000‐fold for fraction II. They were obtained homogenous in disc electrophoresis. These two enzymes have the same molecular weight (about 100 000) and contain one molar equivalent of pyridoxal phosphate. This cofactor is linked as a substituted aldamine derivative (330 nm) at neutral pH values and as an easily dissociable Schiff base in the acid or very basic ranges. An –SH group may be involved in the formation of the aldamine derivative. They differ by their isoelectric point (pI= 5.1 for fraction I and pI= 6.0 for fraction II) and by their number of –SH groups (1 for fraction I and 7 for fraction II). The results suggest that the two synthetases are two isofunctional enzymes.

To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacfer sphaeroides, attempts were made to construct a thioredoxin-def icient mutant by site-specif ic mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TmA-mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R.sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulf ide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in wivo.

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Detergent preparations isolated from thylakoids of the red alga Porphyridium cruentum, in a sucrose, phosphate, citrate, magnesium chloride medium consist of phycobilisomes and possess high rates of photosystem II activity. Characterization of these particles shows that the O2-evolving activity is stable for several hours and the pH optimum is about 6.5 to 7.2. Response of the system to light, electron donors and acceptors, and inhibitors verify that the observed activity, measured both as 02 evolution and 2,6-dichlorophenol-indophenol reduction, is due to photosystem II. Furthermore, photosystem II is functionally coupled to the phycobilisome in this preparation since green light, absorbed by phycobilisomes of P. cruentum, is effective in promoting both 02 evolution and 2,6-dichlorophenol-indophenol reduction. Photosystem II activity declines when light with wavelengths shorter than 665 nm is removed. Both 343,4-dichlorophenyl)-1,1-dimethylurea and atrazine inhibit photosystem II activity in this preparation, indicating that the herbicide binding site is a component of the photosystem II-phycobilisome particle.A PBS3 preparation with PSII activity has been isolated from a red alga, Porphyridium cruentum. in our laboratory has been to ascertain if the PBS is closely associated with the PSII components as expected from energy transfer to PSII (14-16). We had already shown that in a high ionic medium (sucrose-phosphate-citrate) it was possible to retain intact PBS and PSII activity (7). Conditions normally used for PSII preparations (2,4,10,11,13,19,24) result in the loss of PBS by dissociation. We recently reported the successful isolation in SPCM medium, of PIIP particles with high specific rates of PSII activity (1000-3000 ,umol 02* mg:' Chl * h-') from P. cruentum (5). Subsequently, this same medium has been used in the isolation of PSII particles and PBS from a cyanobacterium (17).The PIIP preparation from P. cruentum used in this study is deficient in PSI and has a low Chl content. By electron microscopy the preparations were shown to be discreet particles, consisting primarily of phycobilisomes (6). Thylakoid membranes were extremely rare. In this presentation we show that green light, absorbed by phycoerythrin in the PBS of P. cruentum, is highly effective in driving PSII in PIIP preparations. This demonstrates that PSII does not merely co-isolate with the PBS but is functionally attached. Furthermore, we show that PIIP is a true PSII particle based upon its response to light, inhibitors, and electron donors and acceptors. Inhibitor studies indicate that the herbicide binding protein is a component of the red algal photosynthetic apparatus and is also present in our PIIP preparations. MATERIALS AND METHODS Isolation of Phycobilisomes and PSII-Phycobilisome Particles.Porphyridium cruentum were grown as in Dilworth and Gantt (7) and havested after 7 d growth in the late exponential phase. The PIIP isolation was carried out in SPCM with particle solubilization using lauryl dimethylamine oxide as descr...