Expression, purification, characterization and direct electrochemistry of two HiPIPs from Acidithiobacillus caldus SM-1

High-potential iron-sulfur proteins (HiPIPs) from extremely acidophilic chemolithotrophic non-photosynthetic Acidithiobacillus commonly play a crucial role in ferrous or sulfurous biooxidation. Acidithiobacillus exhibit important industrial applications for bioleaching valuable metals from sulfide ores. In this study, two HiPIP genes from thermophilic Acidithiobacillus caldus SM-1 were cloned and successfully expressed, and their proteins were purified. The proteins displayed a brownish color with an optical absorbance peak at approximately 385 nm and an electronic paramagnetic resonance (EPR) g value of approximately 2.01, which confirmed that the iron-sulfur cluster was correctly inserted into the active site when the proteins were generated in E. coli. The proteins were more thermostable than HiPIPs from mesophilic Acidithiobacillus. The direct electron transfer (DET) between HiPIPs and electrode was achieved by the 2-mercaptopyrimidine (MP) surface-modified gold electrodes; the redox potentials of the HiPIP1 and HiPIP2 measured by cyclic voltammetry were approximately 304.5 mV and 400.5 mV, respectively. The electron transfer rate constant was estimated to be 0.75 s(-1) and 0.66 s(-1), respec-tively. The MP/Au electrode and Au electrode showed consistent differences in heterogeneous electron transfer rates and electron transfer resistances. Bioinformatics and molecular simulations further explained the direct electron transfer between the proteins and surface-modified electrode.