The kinetics of the electron transfer reaction between reduced [2Fe-2S] ferredoxins and select nitroimidazole antimicrobial agents is reported. The ferredoxins from the protozoan Trichomonas vaginalis and the cyanobacterium Anabaena sp. strain 7120 were studied because they are the proximal electron donors to nitroimidazoles in these two organisms with significantly different nitroimidazole susceptibilities. The rates of electron transfer from Anabaena ferredoxin to all nitroimidazoles were 1 to 2 orders of magnitude lower than for T. vaginalis ferredoxin. Quantitative structure-activity analysis of the kinetic data showed that the size of the alkyl substituent on the N-1 position of the imidazole ring strongly influenced the magnitude of the electron transfer rate constant. This implies that the distance between the iron-sulfur cluster and the nitro group of the imidazole is the critical variable in determining the rate of electron transfer. A correlation between the magnitude of the one-electron transfer rate constant with the susceptibility of the host organism to the cytotoxic effects of nitroimidazoles was also discovered. These results demonstrate that reductive activation is the most crucial step in determining the toxicity of nitroimidazoles. (2)] is a member of the nitroheterocycle class of antimicrobial compounds that also includes the nitroimidazole tinidazole and the nitrofuran nitrofurantoin. Metronidazole is a front-line antibiotic in use against a wide range of infectious species. For example, metronidazole is highly effective in treating trichomoniasis and is equally useful against other protozoal infections, including giardiasis and amebiasis (6). Further, metronidazole is the primary drug used to treat postoperative and other infections with anaerobic bacteria, including members of the genera Bacteroides, Fusobacterium, and Clostridium. In addition metronidazole is a component of the therapeutic regimen to eradicate Helicobacter pylori, which is responsible for the majority of cases of peptic ulcer disease and is a risk factor for gastric cancer (4).The importance of metronidazole in treating various infectious diseases and its unusually broad spectrum of activity have led to investigation of its mechanism of action. Most studies have focused on the activity of nitroimidazoles against the protozoan Trichomonas vaginalis, the causative organism of trichomoniasis in humans. Researchers initially believed that the drug inhibited hydrogenase, an enzyme involved in anaerobic carbohydrate metabolism (6). Further studies showed, however, that the ultimate target of the drug was DNA, and that nitroimidazoles not only inhibited DNA synthesis, but also degraded existing DNA (16). Labeling experiments implicated a radical intermediate as the agent responsible for DNA degradation. Within anaerobic cells nitroimidazoles were found to be reduced to radical anion species with the ability to initiate DNA degradation through abstraction of atoms of the nucleic acid backbone (5). The redox potential of nitroimid...