nitrogenase and cyclopropene

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.

Topics: Binding Sites; Carbon Monoxide; Cyclopropanes; Electrons; Klebsiella pneumoniae; Mutation; Nitrogenase; Oxidation-Reduction

The stereochemistry of reductions catalyzed by nitrogenase in 2H2O has been investigated by using allene, methylacetylene, and cyclopropene as substrates. Deuterium labeling patterns in the reduction products were determined by mass spectroscopy, infrared spectroscopy, 2H-decoupled 220-MHz 1H NMR, and 1H-decoupled 30.7-MHz 2H NMR. Reduction of allene gave pure [2,3-2H2]propene. Reduction of methyl acetylene gave a 1.8:1.0 mixture of [cis- and [trans-1,2-2H2]propene. (Similar reduction of acetylene reportedly gave virtually all [cis-1,2-2H2]ethylene.) Reduction of cyclopropene gave [cis-1,2-2H2]cyclopropane and a mixture of [2H2]propenes. The major propene 2H2 isomers formed were [trans-1,3-2H2]-propene (approximately 2), [cis-1,3-2H2]propene (approximately 1) and [2,3-2H2]propene (approximately 1). Cyclopropene appears to be unique as a nitrogenase substrate in that it simultaneously undergoes parallel reductions, one of which proceeds with high stereoselectivity while the other proceeds with low stereoselectivity. The weakly selective stereochemistry observed in these reductions is not consistent with a completely concerted dual proton-dual electron transfer mechanism. The results provide a basis to probe stereochemical effects in nitrogenase and in biomimetic model systems.

Topics: Alkynes; Cyclopropanes; Magnetic Resonance Spectroscopy; Nitrogenase; Oxidation-Reduction; Spectrophotometry, Infrared; Stereoisomerism; Substrate Specificity