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The enzymic activity of phytoene desaturase in Narcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox-active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone-rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox-active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43-kDa NAD(P)H:quinone oxidoreductase, which is organized as a homodimer (23 +/- 3 kDa/subunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23-kDa protein of the oxygen-evolving complex of photosystem II, but no information about the N-terminal organization of the oxidoreductase could be obtained. During flower development, the steady-state concentration of the corresponding mRNA is up-regulated, indicating a specific function of the gene product in chlorophyll-free chromoplasts.

The genome of Rhodopseudomonas palustris contains five antenna gene clusters, alpha beta a, alpha beta b, alpha beta c, alpha beta d and alpha beta e, which encode the light-harvesting peripheral antenna complex II polypeptides. The isolation and characterisation of the gene which encodes the alpha e and beta e polypeptides are reported. The primary structure of the beta e polypeptide is identical to that of beta b whilst the structure of alpha e is different from the other alpha subunits so far characterised. All five of the gene clusters were transcribed under high-light conditions while under low-light conditions only three were transcribed (alpha beta b, alpha beta d and alpha beta e). Furthermore, Northern-blot analysis showed that the gene clusters encode RNA transcripts of either 500 or 650 nucleotides. Individual members of the gene family showed a differential response in terms of the regulation of abundance of mRNA upon growth under either high-light or low-light intensities. Possible promoter sequences and operator sites upstream of the alpha beta b, alpha beta d and alpha beta e genes were located. Furthermore using puc-lacZ fusions in trans in R. palustris, we were able to examine the positions of the promoter of the gene clusters. The significance of these observations with respect to the regulation, organization and role of the peripheral antenna is discussed.

The gene encoding catalase-peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7-kb SacI-HindIII fragment, containing the catalase-peroxidase gene (cpeA) and its flanking regions were determined. A 1728-bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed. A Shine-Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase-peroxidase of R. capsulatus B10. The amino acid sequence of R. capsulatus catalase-peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase-peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase-peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.

A new procedure for isolation, purification and identification of the three polypeptides of the membrane-bound light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata has been developed. The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion-exchange chromatography. The three major polypeptides isolated by this two-step chromatography were found to be homogenous and identical with the three polypeptides of the light-harvesting complex II, as judged by amino acid analysis and N-terminal sequence determination. Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800-850 complex studied by the same criteria.

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.