Suppression by Nitrate of Enzymatic Reduction of Nitric Oxide

Abstract

Barbaree and Payne ( 1 ) found gas chromatographic methods, known to be reliable for determining the components of specifically prepared mixtures of gases ( 2 ) , useful in the analysis of products of denitrification carried out by Pseudomoms perjectomorinuscells and extracts. Employing these procedures, we can now demonstrate routinely that nitric and nitrous oxides (as well as nitrogen) are products of nitrate dissimilation by enzymes in cell-free extracts of this bacterium. I t has been generally known for some time, however, that in growing cultures of denitrifiers the flow of reduction is delayed. Before the oxides and nitrogen are released, nitrite accumulates nearly stoichiometrically with the quantity of nitrate reduced ( 3 ) . Only after nitrate is depleted does dissimilative nitrate reduction go rapidly to completion (e.g., release of gaseous nitrogen). This blockage occurs in P. perfectomarinus as in other denitrifiers and might be explained either (i) by a delay in induction of enzymes for reduotion of nitrate and the intermediate nitrogenous oxides until nitrate is removed, or (ii) by suppression by nitrate of the functioning of the reductive enzymes further along the pathway. The purpose of this paper is to present results supporting the latter explanation.Materials and Methods. We cultured P. perfectomarinus as described by Rhodes et al. (4) in tryptone-yeast extract-sea salt medium containing nitrate (TYSN) . Cells were harvested by centrifugation after 20 hr and washed twice in 0.052 M MgClP, pH 7.0.From these we prepared cell-free extracts as before ( 1 ) and fractionated with ammonium sulfate. The precipitate from 40 to 70% saturation of the extract contained all the dentrifying enzymes. This crude fraction was dialyzed against 0.02 M phosphate buffer, pH Isupported by ONR Grant No. 3677(01) and NIGMS Training Grant 1 TO1 GM01968-01.7.0, for 12 hr with three evenly spaced changes of buffer and then assayed. For certain experiments, the dialyzed fraction was loaded onto a diethylaminoethyl cellulose (DEAE, Whatman DE-52) column, 2.5 X 3.5 cm, and discontinuous elution gradients were employed further to fractionate the protein.We concentrated the column eluates with an Amicon model 50 ultrafiltration cell (UM-10 membrane) before assaying for activity. Protein contents of the extracts were measured by the biuret method (5) and by absorption a t 280 nm. Gas chromatographic analyses were performed as described by Barbaree and Payne ( 1 ) .Release of nitrogen and the nitrogenous gases detectable by gas chromatography was accomplished by supplying the following components: dialyzed extract or eluted fraction as source of enzymes, reduced nicotinamide adenine dinucleotide (NADH) as the electron donor, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as cofactors, and the appropriate oxide of nitrogen as the terminal electron acceptor (6). Before initiation of reactions by the addition of NADH and the appropriate terminal electron acceptor, we sparged the reaction mixtures with helium t...

DISSIMILATORY REDUCTION OF 15N-LABELED NITRATE IN THE PRESENCE OF NONLABELED NO OR N2O

“…The length of this lag period was 5-6 days and was independent of the initial nitrate concentration (Table 3). This last observation is not in agreement with the findings by Payne and Riley (1969) To assess the reliability of the analytical procedures employed in this study, a balance sheet was drawn up for the labeled nitrogen introduced into the two experimental systems. The analyses of recovery were carried out at the termination of the incubation experiments.…”

Effect of pH and Temperature on Denitrification Gene Expression and Activity in Pseudomonas mandelii

“…A separate study investigating the accumulation of denitrification intermediates in Pseudomonas alcaligenes and Pseudomonas fluorescens concluded that nitrite accumulation was dependent on the relative rates of nitrate and nitrite reduction (2). Nitrite accumulation may also be attributed to a lag in the synthesis of nitrite reductase (23,30) or nitrate inhibition of NO reductase (17). In our study, it was hypothesized that enzyme activity was affected over a pH range of 6 to 8, thus causing differences in NO 3 Ϫ utilization and NO 2 Ϫ accumulation among pH treatments over time.…”

Kinetics of denitrification by Pseudomonas denitrificans under growth conditions limited by carbon and/or nitrate or nitrite

“…Nitrate has also been reported to affect the activity of the other enzymes used in denitrification, that is, NOR and nitrous oxide reductase (N20R). Payne and Riley (1969), studying the effect of nitrate on the specific reducing activity of fractions separated from crude extracts of a Pseudomonas bacterium, reported that the presence of nitrate inhibited the rate of enzymatic reduction of NO by the nitric oxide reducing fraction and caused accumulation of N02 -. The suppressive influence of nitrate was curvilinear from 0 to I mmollL at which concentration the activity of NOR was diminished by 60%.…”