Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells

Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.

* Corresponding authors e-mail: sylvain.pichat@ens-lyon1.fr and philippe.oger@insa-lyon.fr The supplementary information includes: Table S1. Calculated average relative Cu concentrations and Cu isotopic compositions Table S2. Isotopic composition measurement of the same S. cerevisiae sample aliquot during four runs over the time course of 3 months  Table S3. Logarithm of the reduced partition function, lnβ (‰), for the pair 65 Cu-63 Cu of Cu(I)amino acid complexes with a description of the computational methods. Table S1. Calculated net Cu concentrations and Cu isotopic compositions (see text as well as (1) and (2) (1) The net Cu concentration reported here is: Cu(t) -Cu(t=0), normalized to 10 6 cells (see Materials and methods for details).
(3) number of replicate measurements of the Cu isotopic composition for each culture replicate experiment, e.g. "4 / 3" means that 4 replicate measurements were made on the first replicate experiment and 3 on the second replicate experiment.
(4) standard deviation (S.D.) between the replicate measurements made for each replicate experiment.  amino acid complexes. Computational methods are given below.

Computational methods
Orbital geometries and vibrational frequencies of aqueous Cu(I) species were computed using the density functional theory (DFT) as implemented by the Gaussian09 code 1-2 . The DFT method employed here is a hybrid density functional consisting of Becke's three-parameter non-local hybrid exchange potential (B3) 3 with Lee et al. (LYP) 4 non-local functionals. Using the 6-311+G(d,p) basis set or higher is recommended for calculating the Cu complexes by de Bruin et al. 5 . The 6-311+G(d,p) basis set, which is an all-electron basis set, was therefore chosen for H, C, N, O, S, and Cu. Molecules were modeled without any forced symmetry. An "ultrafine" numerical integration grid was used and the SCF (self-consistent field) convergence criterion was set to 10 −8 .
The isotope enrichment factor due to intramolecular vibrations can then be evaluated from the reduced partition function ratio 6 in which  stands for vibrational frequency, s for the symmetry number of the Cu compound, h the Plank constant, k the Boltzmann constant, and T the absolute temperature. The subscript i denotes the i th mode of molecular vibration, and primed variables refer to the light isotopologue. The isotope enrichment factor due to molecular vibrations can be evaluated from the frequencies () summed over all the different modes.