The incorporation of p-hydroxybenzoate and isopentenyl pyrophosphate into polyprenylphenol precursors of ubiquinone by broken cell preparations of Rhodospirillum rubrum

Broken cell preparations of Rhodospirillum rubrum have been obtained by subjecting the cells to the combined action of lysozyme and osmotic shock. These lysed preparations incorporate radioactivity from p-hydroxybenzoate-U-14C and acetate-2-14 C into 2-polyprenylphenols and ubiquinones. They also incorporate methionine(methyl-14C) and S-adenosylmethionine-(methyl-14C) into ubiquinones and 2-decaprenyl-6-methoxyphenol. Relatively cell-free extracts were also obtained by sonicating Rhodospirillum rubrum cells. These extracts were efficient in incorporating p-hydroxybenzoate-U-14C into 2-polyprenylphenols. Although ubiquinone synthesis was occasionally observed in both preparations it could be accounted for by the presence of small numbers of unbroken cells in the extract which were detected by means of viable cell counts. These cells were not sufficient to account for the vast majority of 2-polyprenylphenol synthesis and were virtually absent from sonicates capable of synthesizing substantial amounts of 2-polyprenylphenol after centrifugation for 1 hr at 48,000g. The incorporation of p-hydroxybenzoate-U-14C into 2-polyprenylphenol by both types of extract is greatly stimulated by the addition of a source of side-chain precursor in the form of a crude extract of Micrococcus lysodeikticus cells and isopentenyl pyrophosphate. Cysteine and ascorbic acid stimulate this conversion of p-hydroxybenzoate to 2-polyprenylphenol. Attempts to stimulate ubiquinone synthesis in the broken cell preparations by the use of various redox reagents (dichlorobenzenoneindophenol, phenazine methosulfate, methylene blue, neotetrazolium chloride, triphenyltetrazolium bromide, cytochrome c) either in catalytic quantities or as terminal electron acceptors, failed to restore ubiquinone synthesis; most of the reagents proved to be extremely inhibitory to 2-polyprenylphenol synthesis. The R. rubrum extracts, especially when supplemented with Micrococcus extract and isopentenyl pyrophosphate, incorporate radioactivity from p-hydroxybenzoate-U-14 C into another intermediate in ubiquinone synthesis. p-Hydroxybenzoate-7-14 C is also incorporated into this compound, but not methionine-(methyl-14C). The sonicated preparations of R. rubrum are capable of converting this compound to 2-polyprenylphenol. On this basis a reasonable structure to assign to this compound is 3-polyprenyl-4-hydroxybenzoate. A bacterium, found as a contaminant in some cultures of R. rubrum, has been isolated and shown to incorporate phydroxybenzoate-U-14 C into ubiquinone. The organism, when incubated anaerobically with phydroxybenzoate-U-14 C (or p-hydroxybenzoate-7-14C) in the presence of potassium ferricyanide, accumulated 3-polyprenyl-4-hydroxybenzoate. When the cells were washed and the p-hydroxybenzoate-U-14C was diluted with unlabeled p-hydroxybenzoate, subsequent aerobic incubation of the cells converted almost all the 3-polyprenyl-4-hydroxybenzoate to ubiquinone. The distribution of prenylogues of 2-polyprenylphenol in R. rubrum extracts supplemented with MLE and IPP is the same as that observed in whole cells.