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Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

ABSTRACT: Microcoulometric titrations of NADH:nitrate reductase at 25 degrees C in Mops buffer, pH 7.0, showed that the native enzyme, containing functional FAD, haem and Mo, required addition of five electrons for complete reduction. Reduction of the native enzyme occurred in three waves corresponding to addition of reducing equivalents to the centres in the order: Mo, haem, FAD. Oxidation-reduction midpoint potentials (E'0) for the various redox couples were calculated to be as follows: MoVI/MoV, +16 mV; MoV/MoIV, -27 mV; haemoxidized/haemreduced, -172 mV; FAD/FADH2, -283 mV. The values for the haem and flavin are in excellent agreement with those obtained by visible titrations, namely -164 mV and -288 mV respectively. In contrast, the results for the Mo centre are 28-50 mV more positive than the values previously determined by e.p.r. analysis of frozen enzyme samples poised at defined potentials at 25 degrees C and suggest different pH-dependencies or entropies of reduction for the Mo couples.

SUBMITTER: Spence JT

PROVIDER: S-EPMC1148944 | biostudies-other | 1988 Mar

REPOSITORIES: biostudies-other

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Project description:Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases.

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Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

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