PubMed · 1147993
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F M Miesowicz, K E Bloch. 1975-07-08. COENZYME A.. https://doi.org/10.1016/s0006-291x(75)80097-0
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We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.
Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.
1,1,1-Trichloroethane (TCE) is a widely used industrial solvent of low acute toxicity. It is slowly oxidized to trichloroethanol and trichloroacetic acid by cytochrome P-450-dependent mono-oxygenases. Increased inhalative uptake by rats under hypoxia and spin-trapping experiments indicate that TCE is also reductively metabolized to a radical intermediate. Acetylene is formed as a metabolite, suggesting transfer of an additional electron to form the corresponding carbene. Hypoxia and induction of mixed-function mono-oxygenases accelerate the formation of acetylene. Experiments performed in vitro with rat liver microsomal fractions yield analogous results.