Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803.

Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp. PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T. W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I. R., Bryant, D. A., Jones, A. D., Golbeck, J. H., and Chitnis, P. R. (2000) J. Biol. Chem. 275, 8523-8530. We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio. PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ. The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action. The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ. Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site. Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact. Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3. Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group. These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells.

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.