Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia

Abstract

Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth's ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ 15 N) of newly fixed N is currently believed to be ∼-1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)-and iron (Fe)-only "alternative" nitrogenases produce fixed N with significantly lower δ 15 N (-6 to -7‰). An important contribution of alternative nitrogenases to N 2 fixation provides a simple explanation for the anomalously low δ 15 N (<-2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen. Mo is ∼+2‰ (3-6)], which is thought to be the most abundant in nature. As a result, the δ 15 N of newly fixed N is ∼-1‰ (i.e., ∼+2‰ lower than the δ 15 N of dissolved N 2 substrate, +0.7‰, Fig. 1A).In addition to Mo-nitrogenase (the most common form of the enzyme), diazotrophs can possess two other nitrogenase isozymes (7). These so-called "alternative" nitrogenases differ chiefly from Mo-nitrogenases in that V or Fe replaces Mo in the active site. They also contain an additional protein subunit and exhibit slower kinetics compared with the Mo-nitrogenase (8). Such differences could result in distinct isotope effects, a possibility supported by Rowell et al. (9), who reported small but significant variations in biomass δ 15 N from growth of wild-type diazotrophs possessing all three isozymes in media containing Fe and amendments of Mo, V, or neither metal. However, the use of multiple nitrogenase isozymes in the wild type (10) precludes the direct association between biomass δ 15 N and the isotope effect of a particular isozyme. Here we (i) measured directly the isotope effects for Mo-, V-, and Fe-only nitrogenases using diazotroph mutant strains that could express only a single nitrogenase isozyme, (ii) determined the impact of metal limitation on alternative nitrogenase use and N isotope fractionation in wild-type bacteria, and (iii) provide several examples of how these results on N isotope fractionation may change our understanding of the N cycle in the past. Results and DiscussionIsotope Fractionation During Nitrogen Fixation by Mo-, V-, and Fe-only Nitrogenases. We measured the in vivo isotope effect associated with each type of nitrogenase in two phylogenetically and metabolically distinct diazotrophic bacteria, Rhodopseudomonas palustris and Azotobacter vinelandii. R. palustris is an alpha-proteobacterium. It fixes N 2 anaerobically and was grown under anaerobic and photoheterotrophic conditions. A. vinelandii is a gamma-proteobacterium. It fixes N 2 aerobically and was grown under aerobic chemoheterotrophic conditions. All three nitroge...

The evolution of Earth's biogeochemical nitrogen cycle

“…The global mean from four different marine sections from 3.3-2.8 Ga is +1.1 ± 1.9‰ (n = 46) Yamaguchi, 2002). Most of these data points fall well within the range of Mo-based biological N 2 fixation (-2‰ to +1‰, Zhang et al, 2014), especially if possible metamorphic alteration of rocks at greenschist facies (+2.2 ± 1.9‰, n = 26) is taken into account. If correct, this conclusion would imply that alternative nitrogenases, which use Fe (Anf) or V (Vnf) instead of Mo and which cause larger isotopic fractionations up to -8‰ (Zhang et al, 2014), were either insignificant or had not yet evolved.…”

“…Biological nitrogen fixation (diazotrophy; reduction of N 2 to NH 4 + , Table 1) with the most common Mo-based nitrogenase enzyme imparts a small fractionation of -1‰ on average with a range from -2‰ to +1‰, except under Fe 2+ rich conditions or in thermophilic cultures where it can be as large as -4‰ Zerkle et al, 2008;Zhang et al, 2014). Alternative nitrogenases containing V or Fe instead of Mo are less efficient and impart fractionations of -6‰ to -8‰ (Zhang et al, 2014); there is so far no evidence of biological N 2 fixation by alternative nitrogenases in the Precambrian δ 15 N record (Section 4).…”

“…Alternative nitrogenases containing V or Fe instead of Mo are less efficient and impart fractionations of -6‰ to -8‰ (Zhang et al, 2014); there is so far no evidence of biological N 2 fixation by alternative nitrogenases in the Precambrian δ 15 N record (Section 4). Diagenetic remineralization of organic-bound ammonium to dissolved NH 4 + renders residual biomass isotopically heavier by 1.4-2.3‰ under oxic conditions (Freudenthal et al, 2001;Lehman et al, 2002;Moebius, 2013), but under anoxic conditions the effect is probably much smaller and maybe even reversed if light nitrogen is added by anaerobic diazotrophs (Lehman et al, 2002;Robinson et al, 2012) (Section 3.2).…”

“…In an anoxic environment where fixed nitrogen is scarce and Mo-based nitrogen fixation is the major mode of nitrogen acquisition, one would thus expect δ 15 N values between -2‰ and +1‰ in carbonaceous sediments. The Black Sea is a modern example (Fulton et al, 2012); anoxic events in the Cretaceous oceans with values down to -7‰ may represent cases where alternative nitrogenases were dominant (Zhang et al, 2014).…”

“…In rocks between 3.8 Gyr and 3.5 Gyr, negative values down to -4.4‰ (Fig. 6, 7) have been interpreted as representing hydrothermal NH 4 + assimilation by thermophilic microbes (Pinti et al, 2001;Pinti et al, 2009), metasomatic alteration (Li et al, 2014;van Zuilen et al, 2005), or biological N 2 fixation with alternative V or Fe nitrogenases (Zhang et al, 2014). Positive values up to +12.2‰ from rocks of similar age are most commonly interpreted as reflecting metamorphic overprinting, which is known to increase δ 15 N (Papineau et al, 2005;Thomazo and Papineau, 2013).…”