The 15 N-labeled cisplatin, cis-[PtCl 2 ( 15 NH 3 ) 2 ] was synthesized according to the literature method. [1] Protein expression and purification The genes encoding Atox1 was amplified via PCR from a human cDNA library and inserted into pST-SG1 vector using the ligation-independent cloning method. The resulting plasmid was transformed into E. coli BL21(DE3) Gold. Cells were grown at 37 °C in LB medium until the OD 600 of 0.8 was reached, followed by incubation with 0.8 mM isopropyl

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... alactopyranoside (IPTG) for 4 h. Cells were harvested by centrifugation at 4000 rpm at 18 °C for 20 min. The protein was purified using Ni 2+ affinity chromatography. The (His) 6 tag was removed by the TEV protease digestion. Protein was further purified through gel filtration on Superdex 75 16/60 column (GE Healthcare). NMR spectroscopy 15 N-labeled cisplatin were dissolved in Milli-Q water to 3 mM and then lyophilized. Protein samples were prepared in 50 mM sodium phosphate (pH 7.0) containing 10% (v/v) D 2 O and 5-fold dithiothreitol (DTT). For the preparation of Cu I -Atox1 samples, equimolar amounts of Cu(I) complex [Cu(CH 3 CN) 4 ] + was added to apo-Atox1 in the buffer containing DTT. The NMR samples were prepared prior to NMR experiments by mixing protein solutions with the lyophilized cisplatin to give a final concentration of 0.8 mM for both the protein and cisplatin. 1 H-15 N HSQC spectra were recorded immediately to monitor the reaction. NMR spectra were recorded on a 700 MHz Varian Inova spectrometer at 25 °C. The spectral widths were set as 8 ppm ( 1 H) and 60 ppm ( 15 N, centered at -60 ppm). The sequence was optimized with a delay 1/(4J NH ) of 3.42 ms. 15 N chemical shifts of cisplatin were referred to 15 NH 4 Cl. Electronic Supplementary Material (ESI) for Chemical Communications This journal is