Biomedical subjects

J Goretski

Publications and source records attributed to J Goretski.

4 recordsLinked to original sources

PubMed1991

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide.

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.

Alcaligenes↗

PubMed1990

Steady-state nitric oxide concentrations during denitrification.

Three species of denitrifying bacteria, Paracoccus denitrificans, Pseudomonas stutzeri strain JM300, and Achromobacter cycloclastes, were allowed to reduce nitrate or nitrite in anaerobic, closed vials while the equilibration of gases between aqueous and gas phases was facilitated by vigorous stirring. The gas phase was sampled and analyzed for NO with use of a chemiluminescence detector calibrated against bottled NO standards or against NO produced by the nitrite-iodide reaction. [NOaq] was inferred from [NOg] and the solubility of NO. NO was detected only during denitrification in amounts that, once established, did not change with time, were independent of the initial concentration of nitrate or nitrite, and were largely independent of cell concentration, at least when nitrate was the oxidant. The usual level of NO was promptly re-established following the addition of exogenous NO or following the loss of NO by sparging. The aforementioned properties are expected for a steady-state intermediate in denitrification. Steady-state [NOaq] ranged between 1 and 65 nM depending on species and conditions. Similar results were also obtained in a related experiment in which P. stutzeri strain ZoBell respired nitrite under growth conditions. The very low steady-state [NOaq] observed during denitrification imply that the maximum activity of nitric oxide reductase in vivo, if it could be realized, would be large relative to that for nitrite reductase. This circumstance allows NO to be an intermediate without reaching toxic steady-state levels.

Alcaligenes↗

PubMed1990

The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification.

Rates of NO uptake by five denitrifying bacteria were estimated by NO-electrode and gas chromatography methods under conditions of rather low cell densities and [NOaq]. The rates so measured, VmaxNO, represent lower limits for the true value of that parameter, but nevertheless exceed Vmax for nitrite uptake, VmaxNi, by a factor of two typically. Previous estimates under suboptimal conditions had placed VmaxNO at 0.3-0.5 of VmaxNi (St. John, R. T., and Hollocher, T. C. (1977) J. Biol. Chem. 252, 212-218; Garber, E. A. E., and Hollocher, T.C. (1981) J. Biol. Chem. 256, 5459-5465). The steady-state [NOaq] during denitrification of nitrite by nitrate-grown cells was less than or equal to 1 microM. The above observations, taken with a recent direct estimate for the KmNO for NO uptake of 0.4 microM (Zafiriou, O. C., Hanley, Q. S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699), would allow NO to be a free intermediate between nitrite and N2O with steady-state concentrations of less than or equal to 0.4 microM. As the result of special conditions during cell growth or differential inhibition by azide, it was possible to establish systems that accumulated NO during denitrification of nitrite. In all such cases, VmaxNO less than VmaxNi, and the time required to reach the maximum [NOaq] corresponded closely to the time needed to exhaust the nitrite. A semiquantitative isotope experiment with Paracoccus denitrificans demonstrated the trapping of 15NO from 15NO2- in a pool of NOaq. A quantitative isotope method using low densities of the same bacterium showed that 15N from 15NO2- and 14N from NOg combine randomly to form N2O during the simultaneous denitrification of 15NO2- and NO. The result requires that the pathways from nitrite and NO share a common mononitrogen intermediate. Results to the contrary obtained at high cell densities (first two references cited above) are now believed to have been due to technical artifacts. The present results are consistent with the view that NO is under kinetic control as a free intermediate in denitrification and serve to remove previously imagined constraints on this view.

Bacteria↗

PubMed1988

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin.

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.

Antimycin A↗