Expression,Purification,Characterization,and Direct Electrochemical Study Of High-potential Iron-sulfur Proteins From Acidithiobacilus Spp.

High potential iron-sulfur proteins（Hi PIPs）are a class of small molecular weight iron-sulfur proteins containing[Fe₄S₄]clusters with high redox potential.Hi PIPs play important roles in iron-sulfur metabolism of metallurgical microorganism.However,the research on Hi PIPs from Acidithiobacilus spp.was limited to A.ferrooxidans.The exploration of Hi PIPs from other strains of this genus with high application value was not started.Meanwhile,the studies that have been carried out on Hi PIPs were not comprehensive.Direct protein electrochemistry is the most direct way to obtain the oxygen-reduction potential of Hi PIPs,but there are challenges to achieve direct protein electron transfer（DET）.The effective direct electrochemical method needs to be explored.In this paper,we conducted a related study on Hi PIPs from different species of Acidithiobacilus spp.Iro from both A.ferrivorans and A.ferrooxidans had the spectral characteristics of Hi PIPs.The ferrous oxide rate of Iro-FV and Iro-AF was calculated for the first time by a quasi-level kinetic model,and the results indicated that Iro-FV was more capable of oxidizing ferrous iron.Through the experimental exploration of various electrodes and modifiers,a gold electrode modified by 4,6-dimethyl-2-mercaptopyrimidine（DMMP）was established,and the oxygen reduction potential（E⁰）of Iro-FV and Iro-AF was successfully measured to be 435 m V and 427 m V,respectively.The mechanism of DET was further illustrated by Molecular Dynamics（MD）.Characterization of the Hip-FV and Hip-AF showed that their thermal stability in the presence of[Fe₄S₄]clusters was higher than that of the non-metallurgical proteins.The E⁰ of Hip-FV and Hip-AF was successfully measured for the first time using DMMP/Au electrode at 513.5 m V and534 m V,respectively.The reactions of the Hip on the electrode were surface-controlled quasi-reversible processes,and their Ks was 0.537 s^-1and 0.620 s^-1 at the DMMP/Au electrode,respectively,indicated that the iron-sulfur center could respond well electrochemically with the electrode.Protein spectroscopy and paramagnetic studies of two Hi PIP（Hi PIP1and Hi PIP2）from A.caldus,the only thermotolerant genus of Acidithiobacilus spp.,confirmed that both belong to the Hi PIPs family.The Tm of Hi PIP was 80.87°C and 85.77°C,respectively,which were significantly higher than those of Hi PIPs in medium-and low-temperature metallurgical bacteria.After modification of the gold electrode by the optimal modifier 2-mercaptopyrimidine（MP）,the E⁰ of Hi PIP was successfully determined to be 304.5 m V and 400.5 m V,respectively.Based on the potential difference between them,it could be inferred that they have different order of distribution in the electron transfer chain.The similarities and differences in the properties of Hi PIPs from gene to protein level in different strains were compared by bioinformatics and diversity analysis,and their developmental and evolutionary relationships were elucidated.The results of this paper enriched the information on the diverse functional properties of Hi PIPs family under extreme acidic environment.It will provide a reference for resolving the order of electron transfer distribution of multiple enzymes in complex transfer chains and lay the foundation for revealing the mechanism of bioleaching.