Metal-binding amino acid ligands commonly found in metalloproteins differentially fractionate copper isotopes.

Copper (Cu) is a cofactor in numerous key proteins and, thus, an essential element for life. In biological systems, Cu isotope abundances shift with metabolic and homeostatic state. However, the mechanisms underpinning these isotopic shifts remain poorly understood, hampering use of Cu isotopes as biomarkers. Computational predictions suggest that isotope fractionation occurs when proteins bind Cu, with the magnitude of this effect dependent on the identity and arrangement of the coordinating amino acids. This study sought to constrain equilibrium isotope fractionation values for Cu bound by common amino acids at protein metal-binding sites. Free and bound metal ions were separated via Donnan dialysis using a cation-permeable membrane. Isotope ratios of pre- and post-dialysis solutions were measured by MC-ICP-MS following purification. Sulfur ligands (cysteine) preferentially bound the light isotope (63Cu) relative to water (Δ65Cucomplex-free = - 0.48 ± 0.18‰) while oxygen ligands favored the heavy isotope (65Cu; + 0.26 ± 0.04‰ for glutamate and + 0.16 ± 0.10‰ for aspartate). Binding by nitrogen ligands (histidine) imparted no isotope effect (- 0.01 ± 0.04‰). This experimental work unequivocally demonstrates that amino acids differentially fractionate Cu isotopes and supports the hypothesis that metalloprotein biosynthesis affects the distribution of transition metal isotopes in biological systems.

Effects of tidal flooding on estuarine biogeochemistry: Quantifying flood-driven nitrogen inputs in an urban, lower Chesapeake Bay sub-tributary.

Sea level rise has increased the frequency of tidal flooding even without accompanying precipitation in many coastal areas worldwide. As the tide rises, inundates the landscape, and then recedes, it can transport organic and inorganic matter between terrestrial systems and adjacent aquatic environments. However, the chemical and biological effects of tidal flooding on urban estuarine systems remain poorly constrained. Here, we provide the first extensive quantification of floodwater nutrient concentrations during a tidal flooding event and estimate the nitrogen (N) loading to the Lafayette River, an urban tidal sub-tributary of the lower Chesapeake Bay (USA). To enable the scale of synoptic sampling necessary to accomplish this, we trained citizen-scientist volunteers to collect 190 flood water samples during a perigean spring tide to measure total dissolved N (TDN), dissolved inorganic N (DIN) and phosphate concentrations, and Enterococcus abundance from the retreating ebb tide while using a phone application to measure the extent of tidal inundation. Almost 95% of Enterococcus results had concentrations that exceeded the standard established for recreational waters (104 MPN 100 mL-1). Floodwater dissolved nutrient concentrations were higher than concentrations measured in natural estuarine waters, suggesting floodwater as a source of dissolved nutrients to the estuary. However, only DIN concentrations were statistically higher in floodwater samples than in the estuary. Using a hydrodynamic model to calculate the volume of water inundating the landscape, and the differences between the median DIN concentrations in floodwaters and the estuary, we estimate that 1,145 kg of DIN entered the Lafayette River during this single, blue sky, tidal flooding event. This amount exceeds the annual N load allocation for overland flow established by federal regulations for this segment of the Chesapeake Bay by 30%. Because tidal flooding is projected to increase in the future as sea levels continue to rise, it is crucial we quantify nutrient loading from tidal flooding in order to set realistic water quality restoration targets for tidally influenced water bodies.