The aim of the present review is to summarise the knowledge about newly isolated acetogenins (ACGs) in the last six years. It will also report the total syntheses that have allowed either the confirmation or the revision of some structures, together with the biological activities and mechanism of action of such interesting natural products. In fact, of the 417 isolated compounds reviewed, over 176 have been added during the period from 1998 to 2004.

Natural products from the plants of the family Annonaceae, collectively called Annonaceous acetogenins, are very potent inhibitors of the NADH-ubiquinone reductase (Complex I) activity of mammalian mitochondria. The properties of five of such acetogenins are compared with those of rotenone and piericidin, classical potent inhibitors of Complex I. Rolliniastatin-1 and rolliniastatin-2 are more powerful than piericidin in terms of both their inhibitory constant and the protein-dependence of their titre in bovine submitochondrial particles. These acetogenins could be considered therefore the most potent inhibitors of mammalian Complex I. Squamocin and otivarin also have an inhibitory constant lower than that of piericidin, but display a larger protein-dependence of the titre. Squamocin and otivarin, contrary to the other acetogenins, behave qualitatively like rotenone. Rolliniastatin-2 shows unique properties as its interaction, although mutually exclusive to that of piericidin, appears to be mutually non-exclusive to that of rotenone. It is the first time that a potent inhibitor of Complex I is found not to overlap the active site of rotenone.

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kröger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.

The assay of Complex I activity requires the use of artiikial acceptors, such as short-chain coenzyme Q homologs and analogs, because the physiological quinones, such as CoQ,,, are too insoluble in water to be added as substrates to the assay media. The medical interest raised in the last years on the pathological changes of Complex I activity has focussed on the requirement of easy reliable assays for its analysis. We have undertaken a systematic examination of the assay conditions of Complex I in mitochondrial membranes, using a series of quinones as electron acceptors, particularly the coenzyme Q homologs CoQo, CoQl and CoQ,, and the analogs duroquinone and decylubiquinone. Our findings have pointed out that the most suitable electron acceptor for the NADH:CoQ reductase assay is the homolog CoQ1. The analog DB, commercially available, although yielding a high activity, nevertheless causes some problems for the standardization of the assay conditions.

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