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Background and Aims Rice accounts for around 20% of the calories consumed by humans. Essential nutrients like zinc (Zn) are crucial for rice growth and for populations relying on rice as a staple food. No well-established study method exists. As a result, we a lack a clear picture of the chemical forms of zinc in rice grain. Furthermore, we do not understand the effects of widespread and variable zinc deficiency in soils on the Zn speciation, and to a lesser extent, its concentration, in grain. Methods The composition and Zn speciation of Cambodian rice grain is analyzed using synchrotron-based microprobe X-ray fluorescence (µ-XRF) and extended X-ray absorption fine-structure spectroscopy (EXAFS). We developed a method to quantify Zn species in different complexes based on the coordination numbers of Zn to oxygen and sulfur at characteristic bond lengths. Results Zn levels in brown rice grain ranged between 15-30 mg kg-1 and were not correlated to Zn availability in soils. 72%-90% of Zn in rice grains is present as Zn-phytate, generally not bioavailable, while smaller quantities of Zn are bound as labile nicotianamine complexes, Zn minerals like ZnCO3¬ or thiols. Conclusion Zn speciation in rice grain is affected by Zn deficiency more than previously recognized. A majority of Zn was bound in phytate complexes in rice grain. Zinc phytate complexes were found in higher concentrations and also in higher proportions, in Zn-deficient soils, consistent with increased phytate production under Zn deficiency. Phytates are generally not bioavailable to humans, so low soil Zn fertility may not only impact grain yields, but also decrease the fraction of grain Zn bioavailable to human consumers. The potential impact of abundant Zn-phytate in environments deficient in Zn on human bioavailability and Zn deficiency requires additional research. 

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d 10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn 2+ and Cu + binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn 2+ binding was measured by chelation titrations of Zn 7 MT-3, while Cu + binding was measured by Zn 2+ displacement from Zn 7 MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant ( K ) and the change in enthalpy (Δ H ) and entropy (Δ S ) for these metal ions binding to MT-3. Zn 2+ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn 7 MT-2 with Cu + revealed that both MT isoforms have similar Cu + affinities and binding thermodynamics, indicating that Δ H and Δ S are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu 4 + –thiolate clusters when Cu + displaces Zn 2+ under physiological conditions. Comparison of the Zn 2+ and Cu + binding thermodynamics reveal that enthalpically-favoured Cu + , which forms Cu 4 + –thiolate clusters, displaces the entropically-favoured Zn 2+ . These results provide a detailed thermodynamic analysis of d 10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.