Inhibition of proton-translocating transhydrogenase from photosynthetic bacteria by N,N′-dicyclohexylcarbodiimide

Tracy Palmer, Ross Williams, Nick P. J. Cotton, Christopher M. Thomas, J. Baz Jackson

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    12 Citations (Scopus)


    The effects of NN-dicyclohexylcarbodiimide [(cHxN)2C] on the proton-translocating enzyme, NAD(P) H+-transhydrogenase (H+-Thase), from two species of phototrophic bacteria have been investigated. The polypeptides of H+-Thase from Rhodobacter capsulatus are membrane-associated, requiring detergent to maintain solubility. The enzyme from Rhodospirillum rubrum, however, has a water soluble polypeptide (Th(s)) and a membrane-associated component (Th(m)) which, separately, have no activity but which can be fully reconstituted to give a functional complex.

    Two observations suggest that (cHxN)2C inhibited H+-Thase from both species by modification either close to or at the NADP(H)-binding site on the enzyme: (a) the presence of NADP+ or NADPH caused increased inhibition by (cHxN)2C and (b) after treatment of the purified enzyme from Rb. capsulatus with (cHxN)2C, the release of NADP+ became rate-limiting, as evidenced by a stimulated rate of NADPH-dependent reduction of acetylpyridine adenine dinucleotide by NADH. Experiments in which Th(s) and Th(m) from R. rubrum were separately treated with (cHxN)2C then reconstituted with the complementary, untreated component revealed that the NADP(H)-enhanced modification by (cHxN)2C was confined to Th(m).

    In contrast to some experiments with mitochondrial H+-Thase [Wakabayashi, S. & Hatefi, Y. (1987) Biochem. Int. 15, 667 - 675], there was no protective effect of either NAD+ or NADH on the inhibition by (cHxN)2C of enzyme from photosynthetic bacteria. However, amino acid sequence analysis of proteolytic fragments of Th(s) revealed that the NAD(H)-protectable, (cHxN)2C-reactive glutamate residue in mitochondrial H+-Thase might be replaced by glutamine in R. rubrum.

    Original languageEnglish
    Pages (from-to)663-669
    Number of pages7
    JournalEuropean Journal of Biochemistry
    Issue number3
    Publication statusPublished - 1 Feb 1993


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