Reactions of Cytochrome b1 and Nitrate Reductase in a Preparation Solubilized from Escherichia coli

The reactions of cytochrome b1 and nitrate reductase in an enzyme preparation solubiliz-ed and partially purified from the particulate fraction of Escherichia coli were investigated. The cytochrome b1 reduced by the addition of formate and vitamin K3 was found to be reoxidizable by oxygen, nitrate and chlorate. While the oxidation by oxygen was insensitive to cyanide and azide (the autoxidizability of the cytochrome), these reagents strongly inhi-bited the reaction by nitrate and chlorate suggesting the intervention of nitrate reduc-tase. With chlorate as the oxidant, the de-formation of the absorption spectrum of the cytochrome was observed. The nitrate reduc-tion by formate in the solubilized system was found to require the addition of vitamin K3, but flavins had no effects on this reaction. Since the vitamin is required also for thereduction of cytochrome b1 in this prepara-tion, the involvement of the cytochrome in the nitrate-reducing mechanism was further substantiated. The formate-nitrate reaction was strongly inhibited by 2-heptyl-4-hydroxy-quinoline-N-oxide (HOQNO), but this inhibi-tion was not competitive with respect to vitamin K3. It was spectrophotometrically revealed that HOQNO inhibits the reoxidation of cytochrome bt by nitrate, but not the re-duction by formate plus vitamin K3. In the particulate fraction, the formate-nitrate reduc-tase activity was inhibited much more remark-ably than the formate oxidase activity. Some of the implications of these results are dis-cussed.We wish to thank Dr. S. Taniguchi for a gener-ous gift of 2-heptyl-4-hydoxyquinoline-N-oxide