Resistance to triazine herbicides in higher plants was first observed in 1970. A mutation in the photosystem II reaction center D1 protein at position Ser264 --> Gly is responsible for this resistance. So far, 37 single mutants, 16 double mutants, 5 triple mutants and 5 deletion/insertion mutants in the D1 protein have been obtained by randomly induced and site-directed mutagenesis in cyanobacteria and algae. The influence of these mutations on the binding affinities of different classes of herbicides will be discussed. Because a sufficiently high resolution X-ray structure of photosystem II does not yet exist, the reaction center of purple photosynthetic bacteria, which is homologous to photosystem II, served as a model. In the bacterial reaction center a total of 25 single and 3 double herbicide-resistant mutants have been generated.

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A screen has been performed of possible inhibitors of the quinol oxidation sites of the two terminal oxidases of Escherichia coli, cytochromes bo and bd. Aurachin C and its analogues were found to be particularly effective inhibitors of both enzymes, whereas aurachin D and its analogues displayed a selectivity for inhibition of cytochrome bd. In addition, a tridecyl derivative of stigmatellin was found to inhibit cytochrome bo at concentrations which were without significant effect on cytochrome bd. Titration of membrane-bound cytochromes bo and bd with aurachin C gave an observed dissociation constant in the range of 10(-8) M. A similar observed dissociation constant was determined for aurachin D inhibition of cytochrome bd. For both enzymes, their kinetic behavior during a series of substrate pulses indicates that it is reduction of the enzyme by quinol, and not reaction with oxygen, which is inhibited. It is concluded that the aurachins are powerful inhibitors of the quinol oxidation sites of bacterial cytochromes bo and bd. The effects of aurachin C on cytochrome bo were investigated in more detail. The number of inhibitor binding sites on the purified enzyme was determined by titration to be 0.6 per enzyme. At an inhibitor/oxidase ratio of 1.0, electron donation into the enzyme from added quinol is extremely slow, making it very unlikely that there is more than one quinone-reactive site. Aurachin C caused a potent inhibition of electron donation from a pulse of quinol.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are single subunit respiratory chain enzymes found in plant and fungal mitochondria and in many bacteria. It is unclear how these peripheral membrane proteins interact with their hydrophobic substrate ubiquinone. Known inhibitors of alternative NADH dehydrogenases bind with rather low affinities. We have identified 1-hydroxy-2-dodecyl-4(1H)quinolone as a high affinity inhibitor of alternative NADH dehydrogenase from Yarrowia lipolytica. Using this compound, we have analyzed the bisubstrate and inhibition kinetics for NADH and decylubiquinone. We found that the kinetics of alternative NADH dehydrogenase follow a ping-pong mechanism. This suggests that NADH and the ubiquinone headgroup interact with the same binding pocket in an alternating fashion.Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are respiratory chain enzymes that carry out the same redox reaction as mitochondrial complex I. However, unlike this complicated multi-subunit enzyme, they do not contribute to the proton gradient across the respiratory membrane and are insensitive to complex I inhibitors like rotenone and piericidin A (for an overview see Ref. 1). Alternative NADH dehydrogenases are inhibited by flavones in the micromolar range. Acridones have been demonstrated to inhibit both complex I and alternative enzymes (2).Alternative NADH dehydrogenases are found in the respiratory chains of plants (3), fungi (4 -7), many eubacteria (8, 9), and archaebacteria (10 -12). They consist of a single polypeptide chain that exhibits no obvious transmembrane domains and contains one molecule of FAD with the exception of the archaebacterial enzymes that, presumably as an adaptation to thermic habitats, carry covalently attached FMN instead.In most plants and fungi, multiple isoforms of NADH dehydrogenases are expressed in the same species. The active site of the membrane-associated enzymes may be directed to the external or internal face of the mitochondrial inner membrane. In Saccharomyces cerevisiae, for example, two external (SCNDE1 and SCNDE2) and one internal (SCNDI1) alternative NADH dehydrogenases are found (13). The obligate aerobic yeast Yarrowia lipolytica has only a single external alternative enzyme, YLNDH2 (5). It has been demonstrated that this external enzyme can be transformed into an internal version simply by adding a targeting signal for the mitochondrial matrix (14). This suggests that there are no specific protein interaction partners for its membrane association. It remains unclear how the largely hydrophilic enzyme interacts with its highly hydrophobic substrate, ubiquinone. NADH, the other substrate of alternative NADH dehydrogenases, is highly hydrophilic.Inspection of the YLNDH2 sequence revealed two ␤␣␤-dinucleotide-binding domains that consist of two parallel ␤-strands connected by an ␣-helix (5). The sequence G(X)XGXXG, which marks the connection between the first ␤-strand and the connecting ␣-helix, makes close contact with the diphosphate moi...

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In this work, FTIR difference spectroscopy is used to search for possible binding partners and protonable groups involved in the binding of the quinol to cytochrome bd from Escherichia coli. In addition, the electrochemically induced FTIR difference spectra are compared for preparations of the enzyme isolated from cells grown at different oxygen levels in which the quinone content of the membrane is altered. On this basis, difference signals can be tentatively attributed to the vibrational modes of the different quinones types that are associated with the enzyme depending on growth conditions. Furthermore, vibrational modes due to the redox-dependent reorganization of the protein vary depending on the quinone associated with the isolated enzyme. Of particular interest are the observations that a mode at 1738 cm(-1) is decreased and a mode at 1595 cm(-1) is increased as observed in direct comparison to the data obtained from samples grown anaerobically. These signals indicate a change in the protonation state of an aspartic or glutamic acid. Since these changes are observed when the ubiquinone ratio in the preparation increases, the data provide evidence for the modulation of the binding site by the interacting quinone and the involvement of an acidic group in the binding site. The tentative assignments of the vibrational modes are supported by electrochemically induced FTIR difference spectra of cytochrome bd in the presence of the specific quinone binding site inhibitors heptylhydroxyquinoline-N-oxide (HQNO) or 2-methyl-3-undecylquinolone-4. Whereas HQNO leads to strong shifts in the FTIR redox difference spectrum, 2-methyl-3-undecylquinolone-4 induces a specific shift of a mode at 1635 cm(-1), which likely originates from the displacement of the C=O group of the bound quinone.