Kinetics and mechanism of the reaction of cyanide with molybdenum nitrogenase from Azotobacter vinelandii.

The steady-state kinetic behavior of the six-electron reduction of N2 by nitrogenase is known to differ markedly from the six-electron reduction of cyanide in two ways. First, on extrapolation to infinite concentration of cyanide, the H2 evolution reaction is almost completely suppressed whereas at extrapolated infinite concentration of N2, H2 evolution continues. Second, as the ratio of the Fe protein to the MoFe protein increases, the reduction of N2 is favored over H2 evolution, whereas the reduction of cyanide becomes less favored relative to H2 evolution. We have extended these steady-state experiments with Azotobacter vinelandii nitrogenase to include a third observation, that the six-electron reduction of N2 is favored over H2 evolution at high total protein concentrations whereas cyanide reduction is less favored over H2 evolution at high total protein concentrations. All three steady-state observations can be explained by a model whereby cyanide is proposed to bind to a redox state of the MoFe protein more oxidized than that reactive toward H2 evolution and N2 reduction. To test this model, we have examined the pre-steady-state kinetic behavior of both cyanide reduction by A. vinelandii nitrogenase and cyanide inhibition of total electron flow through nitrogenase. The data show that in the presence or absence of cyanide there is a short lag of 100 ms before H2 is detected, followed by a linear phase of H2 evolution lasting for about 3 s, during which time no effects of cyanide are observable. After 3 s electron flow is finally inhibited by cyanide, and the cyanide reduction product CH4 is finally formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase.

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.

Nitrite, a new substrate for nitrogenase.

We have examined the reactivity of the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2) toward nitrate and nitrite. Nitrate has no effect on H2 evolution or C2H2 reduction by nitrogenase and thus is neither a substrate nor an inhibitor. Nitrite dramatically inhibits H2 evolution. This inhibition has two components, one irreversible and one reversible upon addition of CO. The irreversible inhibition is due to nitrite inactivation of the Fe protein. The rate of this inactivation is greatly enhanced by addition of MgATP, suggesting the [4Fe-4S] cluster is the site of nitrite attack. The reversible inhibition does not represent an inhibition of electron flow but rather a diversion of electrons away from H2 evolution and into the six-electron reduction of nitrite to ammonia. Thus, nitrogenase functions as a nitrite reductase.

The use of lipid emulsion as an intravesical medium to disperse light in the potential treatment of bladder tumors.

An obstacle to satisfactory treatment of early bladder cancer with hematoporphyrin derivative-photoradiation therapy is nonuniform illumination of the bladder mucosa. This study was done to determine the characteristics and attenuation of laser light passing through the dispersion medium. Bladder simulation was achieved with the use of 5 different sizes of round-bottom flasks. Intralipid was the dispersion medium. For each flask, 6 different concentrations of the dispersion medium were used. An Argon ion laser was used to stimulate a dye laser at 630 nm. The laser was directed toward the center of the flasks via a fiberoptic cable and energy concentration at 7 different angles was measured. We concluded that the optimal medium was a 1:100 dilution of Intralipid with water, which produced an almost uniform dispersion of light on the walls of the flask. There is a linear correlation between power as measured at the fiber tip and the amount of radiation detected on the walls of the flasks. After determining optimal concentration of medium, we calculated the amount of time needed for a desired energy (Joules/cm.2) to treat a tumor. This calculation was based on the size of the bladder and the power as measured at the fiber tip. The results indicate it is possible to treat the entire mucosa of the bladder with a uniform dose of photoradiation energy.