Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration byEscherichia coliK-12

Abstract

Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA ؉ had no influence on the more rapid growth of narG ؉ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of ⌽(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that ⌽(napFlacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme. Nitrate (NO 3Ϫ ), which is relatively abundant in many environments, has three functions in bacterial physiology. Nitrate assimilation provides a source of ammonium for biosynthesis (reviewed in reference 29), nitrate respiration generates proton motive force for energy (reviewed in references 4, 21, and 65), and nitrate dissimilation oxidizes excess reducing equivalents (reviewed in reference 36).Membrane-bound nitrate reductase (NarGHI enzyme; nitrate reductase A) employs a redox loop to couple quinol oxidation with proton translocation, thereby generating proton motive force for anaerobic respiration. The Escherichia coli and Paracoccus denitrificans enzymes have been the focus of most biochemical studies (reviewed in references 4, 21, and 65). This enzyme contains Mo-molybdopterin guanine dinucleotide, five iron-sulfur clusters, and diheme cytochrome b 556 . NarGHI enzyme activity is inhibited by azide (N 3 Ϫ ). Enzyme synthesis is maximally induced during anaerobic growth in the presence of nitrate.Periplasmic nitrate reductase (NapABC enzyme; nitrate reductase P) also oxidizes quinol, but it is thought that this enzyme is not a coupling site for proton translocation (reviewed in reference 4). Therefore, this enzyme is responsible for nitrate dissimilati...

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures

“…Furthermore, nitrite accumulation did not match nitrate consumption, indicating that, as observed previously (Sears et al, 1997) have been undertaken in Rhodobacter sp. and Escherichia coli (Darwin et al, 1998;Dobao et al, 1994;Gavira et al, 2002;Liu et al, 1999;Reyes et al, 1998;Stewart et al, 2002;Wang et al, 1999). A notable feature of Nap systems is the variation, both within and between organisms, in the physiological functions, as well as the regulation and expression, of nap.…”

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…This enzyme is expressed when nitrate is used as the final electron acceptor in anaerobic conditions [15], as in the case of Nar from denitrifying organisms. This implies that Nap is used as a respiratory system and, like Nap from Escherichia coli K12 (Ec Nap), nitrate reduction should be coupled to a proton electrochemical gradient generated by using the quinone pool [18,19].…”

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

“…In addition to narGHJI, the Riftia and Tevnia symbiont metagenomes also include the napFDAGHBC operon, encoding the periplasmic dissimilatory nitrate reductase NapABC and associated electron carriers. The symbiotic Nap system shows similarity to the anaerobically induced nap operon of Escherichia coli (E. coli) that might compensate the low efficiency of the NarGHI enzyme under limiting nitrate concentrations (Stewart et al, 2002). Clearly, further research is warranted to determine if in fact the symbionts are able to gain energy from denitrification, and to elucidate the details of this process.…”