Determination of Cellular Copper Content

Samples for determination of total cellular Cu contents were prepared as described earlier^{15 (link)}. Briefly, R. capsulatus strains were grown by respiration in 1 L of enriched MPYE medium prepared with metal-free water (stirred at room temperature with Chelex100 at a concentration of 5 g/L for 1 hour) to an OD₆₃₀ of 0.8–0.9. Cells were harvested by centrifugation and washed three times with metal-free 20 mM Tris-HCl pH 8.0 and once with ice cold metal free water. Cell pellets were lyophilized to complete dryness. A total of 50 mg of dry cell powder per sample was digested in 1 ml trace-metal grade nitric acid (Sigma) at 65 °C. To obtain a corresponding blank, the volume of the cell powder was replaced by milli-Q grade water (ultrapure) and treated the same as the samples. The digested samples were then diluted with milli-Q grade water to a final concentration of 1 mg/ml cell powder. Total metal content was measured by ICP-MS (Nexion 350D, Perkin Elmer equipped with an Element Scientific prepFAST M5 autosampler) using quadruplicate digested samples for each strain.