Enzyme specific isotope effects of the Nap and Nar nitrate reductases

Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O) and thus imparts an isotopic signature on residual pools of nitrate in many environments. Data on the relationship between the resulting isotopic pattern in oxygen versus nitrogen isotopes (18{varepsilon} / 15{varepsilon}) suggests systematic differences exist between marine and terrestrial ecosystems that are not fully understood. DNR can be catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases, and previous work has revealed differences in their 18{varepsilon} / 15{varepsilon} isotopic signatures. In this study, we thus examine the 18{varepsilon} / 15{varepsilon} of six different nitrate-reducing microorganisms that encode Nar, Nap or both enzymes, as well gene deletion mutants of the enzymes catalytic subunits (NarG and NapA) to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of the 18{varepsilon} / 15{varepsilon} fractionation ratios of all examined nitrate reductases form two distinct, non-overlapping peaks centered around a 18{varepsilon} / 15{varepsilon} proportionality of 0.55 and a 18{varepsilon} / 15{varepsilon} proportionality of 0.91, respectively. All Nap reductases studied to date cluster around the lower proportionality (0.55) and none exceed a 18{varepsilon} / 15{varepsilon} proportionality of 0.68. Almost all Nar reductases, on the contrary, cluster tightly around the higher proportionality (0.91) with no values below a 18{varepsilon} / 15{varepsilon} proportionality of 0.84 with the notable exception of the Nar reductases from the genus Bacillus which fall around 0.62 and thus closely resemble the isotopic fingerprints of the Nap reductases. Our findings confirm the existence of two remarkably distinct isotopic end-members in the dissimilatory nitrate reductases that could indeed explain differences in coupled N and O isotope fractionation between marine and terrestrial systems, and almost but not fully match reductase phylogeny.