Histidine Intramolecular Nitrogen Isotope Analysis - A Potential New Tracer of Ecosystem Nitrogen Sources

Abstract:

In nitrogen (N) isotope studies of ecosystem structure, Phenylalanine (Phe) is considered the canonical source amino acid (AA) as its δ¹⁵N value is thought to reflect the δ¹⁵N of N sources. However, the α-amino group, containing Phe’s only N atom, is subject to degradation reactions such as transamination and deamination, which may incur isotope fractionation. The molecular structure of Histidine (His) contains 3 N atoms, two of which are in the imidazole side chain and are synthesized from ATP and glutamine respectively. The side chain is less prone to transamination and deamination and is likely re-used by consuming organisms due to the expensive cost of metabolic synthesis. Thus, we hypothesize that the δ¹⁵N of the His side chain (δ¹⁵N_His-side) retains the δ¹⁵N of the inorganic N source with high fidelity, serving as a new tracer to identify N sources and utilization patterns. Both Phe and His are found at low mol% (< 5%) in sediments, but the higher N content in His makes δ¹⁵N analysis more feasible.

δ¹⁵N_His-side analysis is made possible by a new method in which, after hydrolysis, AAs are first separated and collected by Ion-exchange Chromatography with the His fraction divided in 2. One sub-fraction is oxidized by hypochlorite, converting the α-N to nitrite; and the other one reacted with persulfate, oxidizing all three Ns into nitrate. Both nitrite and nitrate are then converted to nitrous oxide and analyzed using Purge-and-Trap Isotope Ratio Mass Spectrometry. The δ¹⁵N_{His α-N} and δ¹⁵N_Phe are determined using a previously published method while δ¹⁵N_His-side could be calculated by the mass balance equation. We will compare the δ¹⁵N_His-side in cyanobacteria, zooplankton, and sediment with the δ¹⁵N_Phe in the same samples and the δ¹⁵N of their corresponding inorganic N sources. This study will provide new insights into key AA biosynthesis and degradation in biota and natural environments.