Abstract
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.
Highlights
Tinct from the much slower chemical reaction of Hb Shapleigh et al [13] reported recently thatTriton X-100 with KNOz to form methemoglobin and HbNO
The initial rates of HbNO production among the above fivedenitrifiers were in therange 0.1-0.2 pmol of Hb heme x min" X" and were similar, when corrected for the fraction of nitrite nitrogen not captured by Hb, to rates of nitrite uptake obtained denitrificans in the presence of rotenone and antimycin A at pH 6.8,25 "C
Reaction was initiated by injection of KNO, and completed within 2 and 6 min for Pa. denitrifzcans and P. stutzeri, respectively
Summary
Instrument Capabilities-Figs. 1 and 2 demonstrate that the spectrophotometer used was able to generate accurate difference spectra of Hb in optically densesuspensions of bacteria. Values as low as 5-12 ~ L Mhave been reported [3].The time required for Pa. denitrificans in succinate phosphate to exhaust 100 p~ Hb upon addition of 250 p M KNOz at pH 6.8, 6250"C, was abo6u0t026, 57, and55907 s for 2, 1,5a0n0d 0.5 mg of cell protein X ml-', respectively. The initial rates of HbNO production among the above fivedenitrifiers were in therange 0.1-0.2 pmol of Hb heme x min" X (mg of cell protein)" and were similar, when corrected for the fraction of nitrite nitrogen not captured by Hb, to rates of nitrite uptake obtained denitrificans in the presence of rotenone and antimycin A at pH 6.8,25 "C. W.succinogenes grownon nitrate showed neither the cellular nor chemical routes to HbNO (Fig. 5) upon addition of nitrate or nitrite. In these experimentcsonsider the data to be only semiquantitative, they indicate the [Hb'] was 70 FM and conditions were otherwise as indi- that most of the nitrogen not trapped as HbNO appears as cated in thleegend to Fig. 3, C and D.Hb' was rapidly reduced N20
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